doi
stringlengths 3
59
| license
stringclasses 95
values | pmc
stringclasses 0
values | pubmed_id
stringclasses 0
values | subset
stringclasses 1
value | text
stringlengths 301
1.8M
|
|---|---|---|---|---|---|
10.1101/2022.05.12.491637
|
cc-by
| null | null |
openalex
|
Background Shotgun metagenomic sequencing is increasingly popular in taxonomic and resistome-profiling of polymicrobial samples due to its agnostic nature and data versatility. However, caveats include highcost, sequencing depth/sensitivity trade-offs, and challenging bioinformatic deconvolution. Targeted PCR-based profiling optimises sensitivity and cost-effectiveness, but can only identify prespecified targets and may introduce amplification biases. Ultra-high multiplexed PCR is a potential compromise. As no comprehensive comparative evaluation exists, we evaluated performance of each method in taxonomic/resistome-profiling of a well-defined DNA mock sample and seven "realworld" wastewater samples.
Results We tested three sequencing approaches (short-read shotgun metagenomics, Illumina Ampliseq TM ultra-plexed AMR Research Panel, 16S rRNA gene sequencing) with seven bioinformatic pipelines (ResPipe, Illumina DNA Amplicon App, One Codex Metagenomic-/Targeted Loci classification and Ampliseq TM Report, DADA2, and an in-house pipeline for AmpliSeq data [AmpliSeek]). Metagenomics outperformed 16S rRNA gene sequencing in accurately reconstituting a mock taxonomic profile and optimising the identification of diverse wastewater taxa, while 16S rRNA gene sequencing produced more even taxonomic outputs which may be quantitatively inaccurate but enhance detection of low abundance taxa. Shotgun metagenomic and AmpliSeq sequencing performed equally well in profiling AMR genes present in a mock sample, but AmpliSeq identified more genes in more complex, "real-world" samples, likely related to sensitivity of detection at the metagenomic sequencing depth used.
Conclusions A complementary sequencing approach employing 16S rRNA gene or shallow-metagenomic sequencing for taxonomic profiling, and the AmpliSeq AMR panel for high-resolution resistome profiling represents a potential lower-cost alternative to deep shotgun sequencing and may also be more sensitive for the detection of low-prevalence AMR genes. However, our evaluation highlights that both the sequencing and bioinformatics approach used can significantly influence results; for AmpliSeq AMR gene profiling, we developed AmpliSeek which outperformed the other pipelines tested and is open source. Sequencing approach and bioinformatic pipeline should be considered in the context of study goals and sample type, with study-specific validation when feasible.Background Shotgun DNA metagenomic characterisation of polymicrobial samples is increasingly used in both clinical and environmental studies, facilitating agnostic sequencing of all DNA present in a sample and enabling flexible comparisons with reference databases to determine sample composition 1 . For example, the RefSeq 2 and CARD databases 3 can be used for taxonomic and antimicrobial resistance (AMR) gene (i.e. "resistome") profiling respectively. This approach has underpinned multiple studies characterising microbiome-disease associations 4 , evaluating community diversity and anthropogenic impacts 5 , and investigating AMR 6, 7 . There is also growing interest in the use of shotgun metagenomics to profile wastewater for population-level surveillance of AMR (i.e. wastewaterbased epidemiology [WBE]) 8, 9 . However, shotgun metagenomics can be expensive, and bioinformatic deconvolution of the data challenging, especially when using short-read sequencing and trying to characterise the genetic context of specific loci, such as AMR genes 10 . Sequencing depth impacts sensitivity to detect rare targets of interest, and therefore, most studies involve a sequencing depth-sensitivity trade-off where uncommon sequences may lack coverage or be missed completely 10 . Additionally, for many studies, only a fraction of the metagenome (e.g. bacterial sequences) will be of interest, and in this context, much of the wider metagenome represents wasted sequencing effort; for example human DNA in studies analysing clinical samples for pathogen diagnostics 1 . Amplicon-based approaches enrich specific nucleic acid templates during library preparation to increase sensitivity for relevant but less abundant target sequences at reduced cost. A commonly used single target for profiling bacterial communities is the 16S rRNA gene which possesses both highly conserved regions facilitating the use of universal primers, and hypervariable regions which can be used to discriminate between bacterial taxa 11, 12 . Increasingly, targeted ultra-highly multiplexed PCR panels have been developed to enable amplicon-based evaluation of the presence/absence and diversity of specific features of a metagenome associated with a key phenotype, for example AMR gene diversity , or of a subset of organisms associated with disease, for example respiratory viruses. One example is Illumina AmpliSeq TM which can be used with both Illumina-and community-curated panels (primer pools). These large panels enable capture of a broader range of targets than universal primers or multiplex PCR, whilst theoretically optimising sensitivity and sequencing resource, and are attractive to researchers as they come with defined laboratory protocols and user-friendly bioinformatic pipelines. However, despite internal validation, Illumina-curated panels have demonstrated mispriming events leading to false positive calls of targets; the community-curated panels have largely not undergone any internal validation. Additionally, targeted methods may be prone to amplification biases, and by their nature only focus on specific features of the wider metagenome. Differences in reference database curation and mapping approaches between available bioinformatic pipelines may also influence results 16, 17 . Combining targeted approaches such as 16S rRNA gene sequencing and ultra-highly multiplexed PCR/amplicon sequencing of AMR gene targets represents a potential alternative to deep shotgun metagenomics for profiling species and AMR gene diversity in polymicrobial samples. We therefore assessed the performance of shotgun metagenomics, versus 16S rRNA gene sequencing and the AmpliSeq for Illumina Antimicrobial Resistance Research Panel (henceforth "AmpliSeq") in reconstituting the true taxonomic composition and resistome of a well-defined DNA mock microbial community. We also compared several bioinformatics approaches to characterising species and AMR gene profiles, including an in-house approach to AmpliSeq data analysis (AmpliSeek). With performance on the mock community as a reference, we then applied the best approach to seven untreated wastewater samples to quantify performance in the context of "real-world" sample complexity. We aimed to highlight potential limitations of each method and recommend potential use cases.
Methods
DNA mock community preparation and wastewater samples Metagenomic, 16S rRNA gene and AmpliSeq sequencing were all conducted on aliquots from the same mock DNA sample (Fig. 1 ). The mock was prepared by combining the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research Corporation, Irvine, USA) with three bacterial isolate DNA extracts chosen to enrich the mock sample for clinically relevant AMR genes (Table S1 ); these bacteria had been characterised with whole genome sequencing as part of a previous study evaluating human bloodstream infections 18 . Wastewater samples comprised seven metagenomic DNA extracts from wastewater influent collected as part of local surveillance in Oxfordshire, UK, 2019, and stored as pellets at -80C. Metagenomic DNA was extracted using the PowerSoil kit (QIAGEN, Hilden, Germany) according to manufacturer protocols.
Sequencing Shotgun metagenomic sequencing on mock DNA community was conducted by Novogene Co (Beijing, China) on the NovaSeq6000 (Illumina, San Diego, USA), generating 150 bp paired-end (PE) reads with a target sequencing depth of 20 million reads (6Gb). AmpliSeq was conducted using the community-designed Illumina for AmpliSeq AMR Research Panel and the Library PLUS kit with manufacturer guidelines; libraries were pooled and sequenced on the Illumina MiniSeq (150 bp PE reads) with replicate libraries prepared for the mock sample. 16S rRNA gene sequencing utilised 515F-806R primers for library preparation and libraries were sequenced on the Illumina MiSeq (250 bp PE reads) as previously described 19 . Wastewater samples underwent sequencing as described above, however, shotgun sequencing was conducted by the Wellcome Trust Centre for Human Genetics (Oxford, UK) with a depth of ~75 million reads (~23Gb) per sample based on previous deep sequencing and rarefaction analyses demonstrating this was the minimum sequencing depth required to capture most AMR gene diversity in this sample type from the same sampling site 10 .
Software We tested seven bioinformatic pipelines (Fig. 1 , Table S2 ). Two pipelines were compared for metagenomic-based taxonomic profiling (ResPipe v1.4.0 and One Codex Metagenomic classification v8/13/2021), two for 16S rRNA-based taxonomic profiling (DADA2 v1. 16 [assignTaxonomy] and One Codex Targeted Loci classification v4/15/2021), and three for AmpliSeq-based resistome profiling (Illumina BaseSpace DNA Amplicon App v0.7.12, One Codex AmpliSeq Report v1/17/2019 and an inhouse BBTools wrapper pipeline [AmpliSeek] developed as part of this study available at https://github.com/KaibondChau/ampliseek). AmpliSeek performs trimming (BBDuk2) and merging (BBMerge) of reads produced by AmpliSeq sequencing before stringent mapping to AmpliSeq panel target sequences (BBMapSkimmer). Reference databases used by each pipeline are detailed in (Table S2 ). Geneious Prime v2021.2.2 was used for detailed in silico characterisation of the mock AMR profile by visualising the relevant AMR reference sequences and reads mapping to these sequences for each methodological approach (Fig. S1 ).
Taxonomic profile analysis We used two previously described error metrics 20 to quantify differences between theoretical and pipeline-reported distribution of bacterial genera present in the mock sample, a modified mean absolute proportion error (MAPE) based on theoretical vs reported read differences, and Bray-Curtis dissimilarity 21 (BC) which further considers the total richness present. Both MAPE and BC scores range between 0 and 1, where 0 represents identical composition. For ease of interpretation, scores are presented as 1-MAPE and 1-BC, where higher values represent better accuracy. Since no theoretical truth existed when characterising wastewater, diversity metrics were used to compare differences between sequencing-pipeline combinations. Chao1 was used as an estimate of taxonomic richness by representing total count of unique observed taxa. Pielou's evenness was used to assess how similarly represented observed taxa were in abundance estimates (constrained to [0,1] with low values representing unbalanced community estimates where few taxa constitute the majority of abundance). Shannon index was used to quantify both richness and evenness as a measure of overall community complexity where higher values represent increased entropy and therefore complexity. A two-tailed Welch paired t test (allowing for different variances in the two samples) was used to determine significance between differences in diversity metrics.
Sensitivity and specificity calculations for AMR gene detection The mock DNA community was annotated with AMR target sequences present in the metagenomic and AmpliSeq reference databases using 100% identity threshold to determine a "strict" in silico truth. These annotations were compared against pipeline calls using scoring thresholds defined by each pipeline (i.e. thresholds at which AMR targets are called present). For AmpliSeek, scoring thresholds were optimised to maximise sensitivity and specificity based on the "strict" in silico truth using receiver operating characteristic curves and Youden's index 22 (Fig. S2 ). Established stringent scoring thresholds for AMR target presence were used for ResPipe (lateral coverage=1) 10 and the One Codex AmpliSeq Report (coverage >= 85% and identity >= 95%) 23 . Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for AMR target detection were calculated by categorising AMR genes called by each method as true positive, true negative, false positive and false negative, when compared with the in silico truth (see above). By adapting this in silico truth with respect to the reference AMR gene database used for each method, we fairly compared overall methodological performance (i.e. the CARD v3.0.3 database of 2605 sequences for metagenomic AMR gene profiling approaches and the AmpliSeq reference panel of 815 [AmpliSeek] or 814 [One Codex] amplicon sequences which target 478 AMR genes). However, AmpliSeek scoring thresholds were optimised to the mock dataset and therefore AmpliSeek performance is not directly comparable to the DNA amplicon app and One Codex Ampliseq Report where scoring thresholds were not optimised to the mock dataset. Mock DNA sequences were also annotated at 98% and 90% identity thresholds to determine "intermediate" and "relaxed" in silico truth respectively to investigate AmpliSeq false positives, i.e. to include more AMR gene matches in the truth set as present where these were similar to a gene in the reference database.
Results
Taxonomic profiling All bacterial genera present in the mock DNA community were correctly detected by each sequencing method-pipeline combination, but the relative abundance of different genera was variably under-and overestimated for each combination (Fig. 2A ). This was most notable for 16S rRNA gene-based classification of Klebsiella which was underestimated by One Codex Targeted Loci classifier and overestimated by DADA2. Bacillus and Lactobacillus were also inaccurately estimated for One Codex Targeted Loci and Metagenomic estimates respectively. Regardless of pipeline, shotgun metagenomics more accurately reconstituted true taxonomic composition over 16S rRNA gene-sequencing (Fig. 2B ; mean 1-MAPE=0.77 vs 0.54, and mean 1-BC=0.87 vs 0.75). Differences in performance between pipelines within each dataset was small, with ResPipe (1-MAPE:0.79; 1-BC:0.88) marginally outperforming One Codex metagenomic classifier (0.75; 0.86) for shotgun sequencing data, and DADA2 (0.55; 0.77) outperforming the One Codex Targeted Loci classifier 0.52; 0.73) for 16S rRNA gene sequencing data (Fig. 2B ).
AMR gene profiling Annotation of the mock DNA sequences with approach-specific reference databases identified 77 CARD and 43 AmpliSeq target sequences present at 100% similarity. These were all detected with 100% sensitivity by all respective methods/pipelines (Fig. 3A ), except the DNA Amplicon App, which performed poorly (22/43 known AmpliSeq targets detected; 51%). For precision, ResPipe (specificity=0.99; PPV=0.72) performed best, followed by AmpliSeek (0.98; 0.69), DNA Amplicon App (0.98; 0.65) and One Codex AmpliSeq Report (0.94; 0.49) (Fig. 3B, 3C ). However, notably, we found that most false positive hits obtained (67/75; 89%) arose from <=2% nucleotide variation from reference AMR sequences (AmpliSeek [18/19], DNA Amplicon App [12/12] and One Codex AmpliSeq Report [37/44]) (Fig. 4 ). Replicate AmpliSeq libraries produced consistent results with no impact on performance metrics for all pipelines tested (Fig. S3 ). AmpliSeq false positives were investigated by comparing pipeline calls to "intermediate" and "relaxed" mock in silico truth (see Methods). We considered AmpliSeq reference sequences present in the mock with 98-100% identity as target variants (Fig. 4 ) and assessed these separately to account for the limited sequence diversity amongst reference sequences for specific targets. An additional 37 reference sequences were identified in the mock at 98-100% identity. When these target variants were included in overall performance metrics, One Codex AmpliSeq Report retained 100% sensitivity with increased specificity and PPV (0.99; 0.92). However, AmpliSeek and DNA Amplicon App were less able to detect target variants, with overall sensitivities of 0.76 and 0.43 respectively. However, AmpliSeek sensitivity improved to 0.90 (Fig. 4 -grey fill) when including less stringent scoring thresholds (i.e. "potentially detected" calls) (Fig. S1 ).
Real-world wastewater samples
Taxonomic profiles Consistent with findings from our mock microbial community benchmarks, taxonomic profiles of "real-world" wastewater samples differed significantly between 16S rRNA and shotgun sequencing, with large differences in relative abundance and detection of core taxa (Fig. 5 ). For metagenomics, Proteobacteria were estimated as most abundant across all samples, with relative abundance approximately two-fold higher than reported by 16S rRNA gene sequencing. Conversely, relative abundance of phyla detected by 16S rRNA gene sequencing across all samples were reduced in metagenomic estimates (Firmicutes, Bacteriodota) or entirely missed/found at <1% relative abundance (Fusobacteriota, Campylobacteriota). Interestingly, this was not the case for Actinobacteriota which was estimated with higher relative abundance than 16S rRNA gene-based estimates by both shotgun pipelines. 16S rRNA gene-based estimates of phylum abundance were more even, with variable detection of Chloroflexi, Patescibacteria, Planctomycetota and Verrucomicrobiota across samples. Interestingly, Campylobacteriota was not detected using the One Codex Targeted Loci pipeline but reported by DADA2. Alternative faceting to highlight difference on a per-sample basis is provided in (Fig. S4 ).
Figure 5: Relative abundance of phyla for wastewater samples (1-7) faceted by sequencing approach and pipeline combination. Metagenomic approaches consistently reported significantly higher richness (Chao1) than 16S rRNA gene sequencing approaches at all taxonomic ranks tested (p<0.002; mean increase of 1354 genera), with the One Codex Metagenomic Classifier identifying significantly more unique taxa than ResPipe (p<0.001; mean increase of 787 genera) (Fig. 6A ). Conversely, 16S rRNA gene sequencing approaches produced profiles with significantly more even representation of taxa (Pielou's evenness; p<0.001) than metagenomic approaches (Fig. 6B ). Despite higher richness in metagenomic results, overall community complexity (Shannon index) was significantly higher for profiles generated by 16S rRNA gene sequencing than metagenomic approaches (p<0.007); except for ResPipe at the genus level. (Fig. 6C ). Interestingly, One Codex Targeted Loci classifier (16S rRNA gene data) produced taxonomic profiles with higher variability across the wastewater samples than all other sequencing-pipeline combinations (Fig. 6ABC ).
AMR profiles Across all wastewater samples, AmpliSeq One Codex Report identified a total of 3220 AmpliSeq targets (2889 called as present, 331 called as probable) while AmpliSeek identified a total of 2157 (1970 called as present, 187 called as potentially present), and the shotgun metagenomics-ResPipe pipeline identified a total of 526 CARD sequences with lateral coverage of 1 (i.e. 100% nucleotide identity over the full length of the reference sequence). When deduplicated to account for targets shared across wastewater samples and redundant AmpliSeq amplicon design (Fig. S1 ), the AmpliSeq One Codex Report detected the most unique AMR genes (n=367), followed by AmpliSeek (n=300) and shotgun metagenomics-ResPipe (n=132), respectively. The distributions of AmpliSeq targets identified by the One Codex Report and AmpliSeek showed the same ratio of increased present calls as the evaluation of the defined mock (i.e. 1.4-fold more AmpliSeq targets called as present by One Codex than AmpliSeek). Similarly consistent with the methodological comparisons on the mock sample, almost all AMR genes called as present by One Codex but absent by AmpliSeek were hits identified at <100% nucleotide identity (817/826; 99%; median nucleotide identity: 98% [IQR:97-99%]). The nine targets identified as present and at 100% identity by the One Codex pipeline but called absent by AmpliSeek possessed low numbers of mapped reads by One Codex (median of one read mapping only). The single AmpliSeq target not targeted by the One Codex AmpliSeq Report (aph2prime-Ia_NT) was called as present by AmpliSeek across all wastewater samples.
Discussion In this study, we have compared the performance of metagenomic and targeted sequencing and different bioinformatics approaches in accurately reconstructing taxonomic and AMR gene composition of a defined mock microbial community, and a set of polymicrobial "real-world" wastewater samples containing complex microbial communities. We highlight that results are significantly influenced by both the sequencing and bioinformatics approach used, and this is an important consideration for studies using these methods.
Mock community evaluation Metagenomics outperformed targeted 16S rRNA sequencing and more accurately estimated true relative abundance of mock genera regardless of the bioinformatic pipeline utilised. While 16S rRNA gene sequencing successfully captured all mock genera, relative abundances differed significantly from the known, true, composition. This was not unexpected as targeted approaches are subject to primer and amplification biases already known to potentially distort taxonomic profiles , however, the degree of bias has not been previously quantified. This was most apparent for One Codex Targeted Loci results where Klebsiella and Bacillus abundances were respectively under-and overestimated by up to three-fold. Conversely, shotgun metagenomics is less susceptible to these biases, and when combined with statistical methods for normalising abundances (as used by both ResPipe and One Codex Metagenomic Classifier), accurately estimated mock genus distributions. For profiling taxonomy, ResPipe and DADA2 slightly outperformed the One Codex Metagenomic and Targeted Loci classifiers. Since classification methods were similar within each sequencing approach (i.e. k-mer based for shotgun metagenomics and alignment-based for 16S rRNA gene profiling), differences in the reference taxonomic databases are most likely responsible for differences in performance 16 , consistent with previous studies demonstrating classification is dependent on database choice 20, 24 . Almost all metagenomics and AmpliSeq approaches detected all relevant unique AMR genes known to be present in the mock microbial community. However, the DNA Amplicon App performed poorly, reporting no reads mapping to 21 AmpliSeq target sequences known to be present in the mock and reported as present by the One Codex AmpliSeq Report and AmpliSeek. This appears attributable to differences in the read mapping workflow rather than alignment algorithm per se since both One Codex AmpliSeq Report and the DNA Amplicon App use the BWA algorithm to align reads. However, instead of directly aligning reads to the reference AmpliSeq target sequences as performed by One Codex and AmpliSeek, the DNA Amplicon App first aligns reads to a reference database of whole genomes containing the reference AMR genes 27 . We noted that this initial genome-based screen appeared to erroneously filter out reads which would otherwise map directly to sequences present in the AmpliSeq AMR panel. However, full details of the DNA Amplicon App workflow are unclear and detailed evaluation of this pipeline was therefore beyond the scope of this study. For AmpliSeq based methods, when focussing on AMR genes known to be present in the mock with 100% nucleotide sequence identity, AmpliSeek slightly outperformed One Codex AmpliSeq Report as the latter reported more false positives. False positives have been previously described with the AmpliSeq panels and attributed to mispriming events associated with primers erroneously binding to non-target sequences 28 . However, mispriming did not appear to be the main cause in this study since most false positives shared high sequence identity to the reference AMR genes (>98%), indicating that these are more likely related to pipeline-specific differences in calling genes present/absent. In fact, the highly related nature of most false positive calls made by the One Codex AmpliSeq Report would be consistent with the default 2% error BWA mapping threshold used in the pipeline to account for sequencing error. The difficulty in calling presence/absence of related AMR genes where the reference sequences used as markers are very similar is highlighted in our analyses: For example, the amplicon reference sequence for blaFEC (blaFEC_T) shares high nucleotide identity with amplicon reference sequences denoting blaCTX-M-3, blaCTX-M-15 and blaCTX-M-32 (blaCTX-M-3_T.2 [99%], blaCTX-M-15_T.3 [99%] and blaCTX-M-32_T.1 [98%]). Modifying the mapping threshold may avoid these issues since recent work has suggested the median error rate of Illumina MiniSeq sequencing reads may be as low as 0.6% 29 . For AmpliSeek we retained stringent mapping of reads as the default to enable differentiation between highly similar reference markers, guard against mispriming, and call specific alleles with high confidence, but this threshold can be relaxed depending on the users' requirements.
"Real-world" wastewater samples On real-world samples, where the true diversity was much higher than in the mock community, differences in the relative abundance of taxa and the number of unique AMR genes observed between approaches when profiling the mock community were greatly amplified. In these samples, metagenomic approaches identified more unique taxa than 16S rRNA gene profiling at all taxonomic ranks tested. This represents a commonly cited key advantage of shotgun sequencing, where higher richness of bacterial taxa can be reported than for 16S rRNA-based methods , and is particularly important in the characterisation of complex microbial communities 31 . Taxonomic classification is also heavily dependent on the reference database used 17 . In this respect, the One Codex Metagenomic Classifier yielded significantly higher richness than ResPipe likely owing to respective reference species database sizes of ~115,000 and ~20,000 complete genomes. Taxonomic richness reported by ResPipe was also seemingly capped across most ranks with minimal variation between wastewater samples which may be a result of saturating the reference database as previously suggested 10 . Metagenomic abundance estimates were largely skewed towards a small number of taxa; in contrast, 16S rRNA gene-based estimates were consistently more even across all ranks regardless of pipeline, and more sensitive to the detection of taxa missed by shotgun metagenomic outputs. This likely partly reflects the sensitivity trade-off of shotgun sequencing where sequencing effort can be swamped by high abundance taxa and/or taxa with large genomes; especially when sequencing depth is low and community complexity is high 1 , which can result in missing the presence of lowabundance, but significant, taxa 33 . However, users of 16S rRNA gene sequencing for taxonomic profiling need to be aware of the likely greater divergence of profiles from the truth. In our hands, different 16S rRNA gene pipelines also notably impacted results; for example, Campylobacteriota were absent from One Codex outputs but classified as present using DADA2, further highlighting differences across reference databases. Interestingly, despite being more sensitive in profiling AMR targets in the mock, metagenomics detected less than half the number of AMR genes reported by targeted AmpliSeq approaches. This is potentially driven by low abundance AMR genes which are missed by an agnostic approach, but directly targeted and amplified by AmpliSeq. However, we used the strictest definition of AMR gene presence requiring read matches covering all of the reference sequence (lateral coverage=1), so our metagenomic profiling was a conservative estimate. As discussed, sequencing depth impacts the sensitivity of target detection in complex samples, with the relative simplicity of the mock composition (n=11 bacterial genomes, 77 CARD sequences, 43 AmpliSeq sequences) masking this limitation. As seen in the evaluation on the mock, the less stringent mapping thresholds of the One Codex Report produced more present calls than AmpliSeek due to calling target variants as present. In fact, the proportion of increased present calls made by One Codex Report compared to AmpliSeek was consistent between mock results and wastewater results. These findings support similar performance of both AmpliSeq pipelines in wastewater as in the mock despite significantly increased complexity, where AmpliSeek results represent increased confidence in the presence of the exact marker sequence.
Limitations We have not evaluated all methods for profiling taxonomic and resistome composition and therefore potentially missed approaches with increased performance. However, our pragmatic evaluation focussed on accessible approaches (i.e. kit-based and all-in-one pipelines) which are commonly used, attractive to researchers and have not undergone comparative evaluation. The relative simplicity of the mock DNA community may mask sensitivity issues owing to sequencing depth but use of in silico simulated datasets would otherwise omit assessment of library preparation amplification reactions. To mitigate this, we enriched the mock with additional AMR determinants and presented performance differences between the mock and "real-world" samples. Our in-house pipeline AmpliSeek was internally validated on the same mock dataset used for performance metrics which inherently optimises performance, however, the "real-world" samples represented an external validation confirming similar performance with and without scoring threshold optimisation. We also guarded against wastewater metagenomic sensitivity issues by using optimised depth as determined by previous ultradeep pilot sequencing performed on wastewater form the same sampling site 10 . Our resistome characterisation focusses on presence/absence but a major advantage of shotgun metagenomics is quantitative measurement of genes which we have not explored here. Additional factors such as read length and approaches to normalization may also optimise each approach but exploring these were beyond the scope of this study.
Conclusion A complementary sequencing approach encompassing 16S rRNA or shallow-metagenomic sequencing and the AmpliSeq AMR panel represents a potential lower-cost alternative to deep shotgun sequencing for taxonomic and resistome profiling of highly diverse, complex, polymicrobial samples. Accuracy of profiling was also to some extent dependent on the bioinformatics pipeline used; for AmpliSeq AMR gene profiling we have optimised an in-house pipeline (AmpliSeek) which outperformed the other methods tested on this dataset and is open source. However, further validation of this in-house pipeline is needed, since it was also developed on the same dataset. We recommend careful consideration and further validation of sequencing approaches and bioinformatic pipelines in the context of study goals and sample type to be analysed. Validating sequencing and bioinformatic approaches remains challenging due to difficulties in representing the true complexity of "real-world" samples; any conclusions drawn should always be considered in the context of the methods used. Figure 1 : 1 Figure 1: Schematic overview of evaluated sequencing approaches and respective bioinformatic pipelines tested for taxonomic and resistome profiling of the DNA mock.
Figure 2 : 2 Figure 2: (A) Theoretical taxonomic composition of DNA mock as compared to actual generated pipeline outputs faceted by sequencing approach. Genus "Other" represents genera present at <1% relative abundance in pipeline outputs. For the theoretical facet, "Other" represents DNA composition from yeasts included in the commercial DNA standard. (B) Taxonomic profiling error scores represented by 1-BC and 1-MAPE where 1.00 is perfect reconstitution of theoretical genera abundances.
Figure 3 : 3 Figure 3: (A) Detection sensitivity of known AMR genes present in the mock at 100% identity. NB the reference AMR gene database used to annotate the mock was specific to, and hence different for, each approach. Error bars represent 95% confidence intervals (CIs), and facets divide sequencing approach and bioinformatic pipelines. (B) Specificity of AMR gene detection presented as for (A) and with magnified y-axis in (C).
Figure 4 : 4 Figure 4: Heatmap of AmpliSeq pipeline calls labelled by reference marker ID and sequence identity.Each column represents a bioinformatic pipeline output where black and grey fill reflect detected and potentially detected calls respectively. Labels (green, blue, orange) denote the sequence identity of reference markers when annotated in the mock DNA community.
Figure 6 : 6 Figure 6: Distribution of diversity metrics across all wastewater samples coloured by sequencing approach and pipeline combination. Panels: 6A reports richness via log10 Chao1, 6B reports abundance distribution via Pielou's evenness, 6C reports overall alpha diversity via Shannon index.
|
10.35470/2226-4116-2022-11-4-217-226
|
cc-by
| null | null |
openalex
|
We consider one of the cybernetic methods in biology related to the study of DNA chains. Namely, we are considering the problem of reconstructing the distance matrix for DNA chains. Such a matrix is formed on the basis of any of the possible algorithms for determining the distances between DNA chains. The objects of research of these algorithms (for mammals), as a rule, are one of the following 3 variants: the main histocompatibility complex, the mitochondrial DNA, and "the tail" of the Y chromosome. In the paper we give an improved algorithm for restoring the distance matrix for DNA chains. Compared to our previous publications, the following changes have been made to the algorithm. We abandoned the use of the branches and bounds method, but at the same time significantly improved the greedy auxiliary algorithm used in it. In this paper, we apply only this greedy algorithm to the general solution of the distance matrix reconstruction problem. As a result of the conducted computational experiments carried out on one of the two considered criteria for the quality of the algorithms, significant improvements were obtained compared to the results given in our previous publications. At the same time, the total running time of the algorithm remained almost the same as in the previous version.Introduction In this paper, we continue to consider one of the cybernetic methods in biology related to the study of DNA chains. Namely, we are considering one of the important tasks of this topic, i.e., the problem of reconstructing the distance matrix for DNA chains. In this case, the distance matrix is formed on the basis of any of the possible algorithms for determining the distances between DNA chains of monkeys, as well as any specific object of study. "Plants, animals and bacteria all contain the essential biological molecule known as DNA or deoxyribonucleic acid. DNA contains all the information required to build and maintain living organisms. You can think of it as nature's very own top-secret instruction manual . . . " "This manual is written in multiple combinations, but limited to just 4 letters: A, T, G and C. Each letter denotes a nitrogenous base: A for adenine, T for thymine, G for guanine and C for cytosine. Every living being has a huge supply of these 4 bases, each of which is attached to a pentose sugar and a phosphate molecule. Together, they are known as a nucleotide. These nucleotides are arranged in two long coiled strands like a hair braid." "Every single cell which builds up a living organism carries information for various functions necessary for the survival of the cell. This genetic information in each cell is stored in molecules called nucleic acids. The most stable form of nucleic acids is called deoxyribonucleic acid (DNA). Each of the DNA strands forms helical structures that are long polymers of millions of linked nucleotides. These nucleotides consist of one of four nitrogen bases, a five-carbon sugar, and a phosphate group. The nitrogen bases -A (Adenine), T (Thymine), G (Guanine), C (Cytosine) encodes the genetic information . . . " ( https://www.scienceabc.com/pure-sciences and https://whatisdna.net/ ) However let us remark, that the total length of the human genome exceeds 3 10 9 characters, which is about 200 000 times longer than mt DNA (see below). This fact indirectly confirms the need to apply heuristics when considering DNA algorithms. It is important to note that currently it is easy to find only a few similar algorithms on the Internet, [Needleman and Wunsch, 1970; Winkler, 1990; van der Loo, 2014] etc. (the authors' usage of Internet searches give about 10 similar algorithms only); see also the description of our algorithm in [Melnikov, Pivneva, and Trifonov, 2017 ] and some of our other papers cited there. The objects of research of these algorithms (for mammals) are, as a rule, one of the following 3 variants: mt DNA, the mitochondrial DNA, inheritance in the "direct female line", see [Maloy and Kelly (Eds), 2013; Cibelli et al. (Eds) , 2014] etc.; for human, the the length of its sequence exceeds 16 000 characters; "the tail" of the Y chromosome, inheritance in the "direct male line", see [Sykes, 2003, p. 290] etc.; for human, the the length of its sequence exceeds 50 000 characters; MHC, the main (major) histocompatibility complex, see [Lennarz and Lane (Eds) , 2013] etc.; usually, we cannot say about its length. "The structure of MHC allows to bind peptides of varying lengths because both ends of the peptide are free . . . ", see ibid. "The MHC complex encodes the α-chains of the MHC class I molecules human leukocyte antigen (HLA)-A, HLA-B, and HLA-C and the αand β-chains of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ, all of which are expressed in a co-dominant fashion." "MHC class I is expressed by all nucleated cells and platelets in jawed vertebrates, although the amount on the cell surface varies among cell types and under different inflammatory conditions." "The folded MHC class II molecule consists of two transmembrane proteins, an α-chain and a β-chain, which together form a protein . . . Peptides bound by MHC class II molecules typically are longer than 10 amino acids and occasionally more than 20 amino acids." However, such a small number of variants (less than 10 algorithms and 3 objects of research) does not negate the need to create effective algorithms for processing DNA chains, in particular, constructing (for one of these variants) a matrix of distances between such chains. At the same time, the distances between DNA sequences are often used in scientific and popular science literature. However, as we already said, there are several different algorithms for calculating them, and for each pair of sequences, the operation of any of these algorithms takes a lot of time. For example, the practical programming results show that on an average modern computer, it takes about a day to build such a 30 × 30 matrix for mtDNAs using the Needleman -Wunsch algorithm [Needleman and Wunsch, 1970] ; therefore, for such a 300 × 300 matrix, about 3 months of continuous computer operation is expected. Such dimensions come from real problems: for example, in the class of mammals there are about 30 orders, in the order of primates there are about 20 families, more than 80 genera and more than 500 species. At the same time, the exact values differ in different classi-fication options, but they are not interesting to us: we are interested in approximate values only. Thus, even for a relatively small number of species (smaller than the total number of primate species), calculating the distance matrix on conventional computers is hardly feasible; the use of supercomputers, firstly, is not always possible, and, secondly, it often requires significant revision of existing software. In this regard, the task of restoring such a partially filled matrix arises. We started publishing our variants of similar algorithms for restoring partially filled matrices in [Melnikov, Pivneva, and Trifonov, 2017] (the simplest algorithm was described very briefly there), after which we returned to this problem in [Melnikov and Trenina, 2018a; Melnikov and Trenina, 2018b] , where a variant of the algorithm using the method of branches and boundaries was described in detail. Remark. At the same time, it is worth noting that in the last two papers we made computational mistakes, which, however, did not affect the overall assessment of the results of calculations given in these papers at all. For example, it was said that we leave about 30-35 % of the elements in the matrices of dimension about 30 × 30, obtained for processing, while we left a significantly smaller number of elements, i.e., about 10 %; thus, we solved a more difficult task. Certainly, the latter fact indicates much greater possibilities in the application of the algorithms we are considering. In addition, some computational errors were made when obtaining obtaining the values of σ and δ, which, again, did not affect the evaluation of the calculation results. Despite the previously successful results of calculations, we return in this paper to the algorithm variants that do not use the branches and boundaries method: as our recent work has shown (primarily in the subject areas related to graph theory and the development of ultra-large communication networks, [Melnikov and Terentyeva, 2021] etc.), even greater improvement in the quality of the algorithm can often be achieved without improving the auxiliary heuristics of the branches and boundaries method. We are improving the algorithms that formulate the greedy function of this method only; however, these algorithms can also be called auxiliary to the method of branches and boundaries. In this paper, we describe a similar improvement of the greedy algorithm, now for the task of reconstructing the matrix of distances between DNA chains. As the obtained results of computational experiments show (see Section 5), they are better than ones obtained using simple variants of the branches and boundaries method. Let us repeat that for restoring partially filled matrices, i.e., for the inverse problem of matrix processing, we used the method of branches and boundaries before, but in this paper, we do not use it. In connection with the above, the question arises about the concept of "partially filled matrix": how to determine this partial filling. It is clear that the greater the percentage of values will not be calculated, the less time will be spent on these calculations: after all, as follows from the above estimates, the calculation of one value for two considered mt DNA s requires about 3 minutes of computer operation, which is approximately equal to the total time required for matrix recovery even using the long-running method of branches and boundaries. On the other hand, a too small percentage of the values left in the matrix (i.e., calculated by the special previous algorithms), of course, cannot give adequate results; in this regard, we have been using in this work the percentage of values calculated by the algorithm of about 10-12 %. The second important question that cannot but arise on the basis of the above text is how exactly we can analyze the quality of the solution obtained using the recovery algorithm(s). For more information, see Section 5 below. The paper has the following structure. In Section 2, we consider a brief description of the greedy algorithms of restoring the distance matrix. In Section 3, we give the theoretical substantiation of the possibility of improvement of the greedy algorithm (without the variants of the method of branches and boundaries). In Section 4, we formulate two possible quality criteria for the numerical solution of such restoring problems: the first criterion compares the matrix reconstructed by the simplified algorithm under consideration with the matrix obtained as a result of applying a general formation algorithm for each of its elements; and the second criterion considers the discrepancy in a special way, it applies the same algorithms that are used as auxiliary ones of the general recovery algorithm considered in this paper. In both cases, the goal is to reduce the values obtained by the applied criterion. In Section 5, we give some results of computational experiments; we evaluate these results well. In Section 6, we formulate some problems for the future solution. And in Conclusion (Section 7), we briefly repeat the content of this paper. 2 A brief description of the greedy algorithms of restoring the distance matrix It is clear that with smaller dimensions of the matrix, a larger percentage of non-deleted elements is required. Thus, for small dimensions (of the order of 10), algorithms often do not work with a small number of nonremovable elements. Of course, in principle, there are options when, under the conditions we have given (i.e., about 10 % of non-removable elements with matrix dimensions of about 30), it is impossible to restore the matrix: for example, when an empty line is obtained. When calculating using the elementary formulas of probability theory, we find that the probability of this event is about 25 %. However, we simply do not consider such examples as source data. We considered the simplest heuristic for filling in the distance matrix without using the method of branches and boundaries in [Melnikov and Trenina, 2018a] . (We note in advance that the results of the computational experiments given in that paper will be compared below with newer results using other heuristic algorithms.) Further, as we have already noted, our publications were devoted to the application of the branches and boundaries method; but in this paper, we again abandon it. At the same time, we complicate the greedy heuristics of [Melnikov and Trenina, 2018a] . Let us briefly describe the greedy matrix filling algorithm used in this paper. First of all, we choose an element that, if filled in, forms the largest number of newly formed triangles; i.e., for n×n-dimensional distance matrix (m i,j ), we choose the pair (i, j) such that: m i,j < 0 (in our natural notation, this means that there is no corresponding matrix element in the input data); and the following formula is achieved: max 1⩽i,j⩽n i̸ =j 1⩽k⩽n k̸ =i, k̸ =j sgn(m k,i ) + sgn(m k,j ) . If there are several such elements, we choose any of them. Next, we consider all the resulting triangles, and minimize the total value of the badness. One of the variants of such an assessment of badness for one triangle is the formula α -β γ , where α, β and γ are the angles of the derived triangle, and α ⩾ β ⩾ γ. (Note that earlier we sometimes used another formula, a-b c , where a, b and c are the sides of the derived triangle, and a ⩾ b ⩾ c. At the same time, in both cases, if the three sides do not satisfy the triangle inequality, we assumed a large value as the value of badness, usually from 1.0 for the case a=b+c to 2.0 for "absolutely impossible" triangles.) The total value of badness is always (i.e., both for choosing a value in the described algorithm and for a posteriori evaluation of the quality of the algorithm) considered simply as the sum of the values of badness of all triangles. In the described algorithm, we are trying to minimize this badness value for all newly formed triangles. The minimization method is given in the next section, where the justification for the possibility of piecemeal filling of the matrix is given, to obtain a value of badness close to optimal (i.e., in terminology of [Melnikov et al., 2018c] , "to obtain a pseudo-optimal solution"). Simplifying it, we can say that that, taking the average values of the maximum sides of the formed triangles (i.e. α in previous formulas; note that in [Melnikov and Trenina, 2018a] , this value was counted final) as the beginning of the iterative process, we get a pseudo-optimal value in a few iterations. (We usually limited the number of iterations to 10, this gives an acceptable value of the calculation time.) 3 The theoretical substantiation of the possibility of improvement of the greedy algorithm Thus, in this paper we reconstruct the matrix of distances between DNA sequences of different species of organisms. We shall restore the distance function of the matrix and find its derivative. The main problem is that it is an ill-posed problems, which means that a small error in the source data can lead to a large error in the calculated derivatives [Tikhonov, 1963; Groetsch, 1984; Hanke and Scherzer, 2001; Chaikovskii and Zhang, 2022] , etc. If we introduce the coordinate axes x and y, located in the horizontal and vertical axes, respectively, and consider the matrix as a two-dimensional array with noisy data defined on the domain Ω ∈ R 2 , then the matrix elements will represent the noisy values of the function of two variables u δ i,j . It is natural to assume that the domain Ω is divided into N ⩽ n 2 parts {Ω i } N i=1 , and there is the only one value u δ i,j in each parts. Denote d i as the diameter of Ω i and let d = max{d i }. In this case, we obtain the deterministic model max|u(x i , y j ) -u δ i,j | ⩽ δ between the noisy data {u δ i,j } and the corresponding exact values {u(x i , y i )} at grid points X n := {x 1 < x 2 < < x n } and Y n := {y 1 < y 2 < < y n }. Let us suppose that the reconstructed function u ε (x, y) is formed according to the following optimization problem: u ε = arg min v∈C 1 (Ω) 1 n 2 n i=1 n j=1 v(x i , y j ) -u δ i,j 2 + ε ∂ 2 v(x, y) ∂x 2 2 L 2 (Ω) + ∂ 2 v(x, y) ∂y 2 2 L 2 (Ω) , (1) where v(x, y) is a cubic spline, and regularization parameter ε satisfies the expression 1 n 2 n i=1 n j=1 u ε (x i , y j ) -u δ i,j 2 = δ 4 . Then by [Wang and Wei, 2005, Theorem 3 .3], the following proposition holds. Proposition 1. Let u(, ) ∈ H 2 (Ω). Let u ε (x, y) be the minimizer of the problem (1). Then for ε = δ 2 , we have ∥u ε (, ) -u(, )∥ H 1 (Ω) ⩽ C 1 d 1/4 + C 2 √ δ, where C 1 and C 2 are some constants depending on the area Ω and on ∥∆u(x, y)∥ L 2 (Ω) . Based on Proposition 1, we obtain the following fact. For sufficiently small values d and δ, after solving the optimization problem (1) values ∂u ∂x , ∂u ∂y can also be found with sufficiently high accuracy. We can do it taking derivatives ∂u ε ∂x , ∂u ε ∂y . Due to the fact that with an increase in the value of the norm ∥∆u(x, y)∥ L 2 (Ω) the value of constants C 1 and C 2 will increase, we conclude that the smoother the function u(x, y) is, the smaller this norm will be, and the more accurate the regularization result will be. If the function is not smooth enough, we shall need more noisy data to obtain the necessary accuracy. Another smoothing technique can be used is the convolution, see [Gulliksson et al., 2016, Theorem 3 ] and [Lin, Cheng, and Zhang, 2018, Section 4] for details. It also follows from the proposition 1 that if we set δ → 0, the error of restoring the function will depend mainly on the diameter d, which is the larger, the more missing data {u δ i,j } at the grid points. And due to the fact that 65 % of data is missing in the task we have set, we need to introduce additional conditions to restore the function. One of such conditions is the regularity found in the paper [Melnikov, Pivneva, and Trifonov, 2017] for distance matrices, which consists in the fact that the three elements of the matrix (m i,j , m k,j , m k,i ) form the sides of an isosceles triangle. Thus, the formulas below reduce such a metric to a function of several variables, and a "triangular" norm for determining the quality of the distance metric can be introduced, which can be represented in the following way. For the matrix M = (m i,j ) and its elements m i,j , we always 1 suppose that i, j, k ∈ { 1, 2, . . . , n } and do not consider diagonal elements (i.e., elements m i,i and the arithmetical expressions with these elements are ignored in formulas). The total error σ is defined as follows: n-1 i=1 n j=i+1 σ i,j , where one of the calculation variants is the sequential usage of the following formulas: r (1) i,j,k = max m i,j , m k,j , m k,i , r (2) i,j,k = min (m i,j , m k,j , m k,i ), and σ i,j is as follows: max 1⩽k⩽n k̸ =i,k̸ =j 2r (1) i,j,k + r (2) i,j,k -m i,j -m k,j -m k,i r (2) i,j,k . Then the original problem can be reformulated into the problem of minimizing the error value σ (as we already said, it often was called "badness" in our previous papers) by piecemeal filling in the missing elements. It is very important that we fill missing elements in the table sequentially, piecemeal, "step by step"; thereby we greatly simplify the implementation of the corresponding algorithm. Filling the table in this way, we obtain a matrix with noisy data {u δ i,j } and then we restore the u ε function by solving (1). The level of noise δ generated by this algorithm for restoring missing values can be estimated by analyzing the results of violations of the "isosceles triangle" regularity in [Melnikov, Pivneva, and Trifonov, 2017] . Thus, by sequentially filling in the missing elements of the matrix, we can guarantee a consistent improvement of the resulting solution, which theoretically justifies the possibility of abandoning the branch and boundary method, which works much more longer than the greedy algorithm for obtaining the value of one element considered here. 4 Quality criteria for the numerical solution of the problem As we said before, an important question arises, is how exactly can we analyze the quality of the solution obtained using the recovery algorithm(s). However, the above model of calculations does not give a complete answer to the question of the quality of matrix restoring. Therefore, the simplest quality criterion would be a comparison (by some natural metric) of the reconstructed matrix and the actually obtained distance matrix, which we can obtain for some examples of a small dimension; however, it is obvious that such a comparison can be made only a limited number of times, probably during the initial debugging of the algorithms. Therefore, in this section we formulate two possible quality criteria for the numerical solution of such restoring problems: (1) the first criterion compares the matrix reconstructed by the simplified algorithm under consideration with the matrix obtained as a result of applying a general algorithm for the formation of each of its elements, like [Melnikov and Trenina, 2018a; Melnikov and Trenina, 2018b] ; we shall denote by σ the value of this criterion; (2) and the second criterion considers the discrepancy in a special way, it applies the same algorithms that are used as auxiliary ones of the general recovery algorithm considered in this paper; we shall denote by δ the value of this criterion (or by d in some previous papers). In both cases, the goal is to reduce the values obtained by the applied criterion. The exact formulas are as follows. (1) For σ, we usually set σ = 2 n (n-1) n-1 i=1 n j=i+1 (m i,j -m i,j ) , where all the elements m i,j are obtained by applying the original algorithm (for instance, already cited Needleman -Wunsch algorithm), i.e., without restoring any elements. Note that for obvious reasons, we cannot often use this method, and also we cannot apply it for large matrices obtained by some distance determination algorithms; therefore the following criterion δ can be called more universal. (2) For δ, we usually set δ = n-2 i=1 n-1 j=i+1 n k=j+1 δ i,j,k (we specifically note once again, that the values m i,j are not used here). Each value δ i,j,k (where (2b) If a ⩾ b + c (i.e., the triangle inequality is violated 2 ), we choose in advance a constant ω (usually, ω = 2) and set 1 ⩽ i, j, k ⩽ n, i ̸ = j, i ̸ = k, j ̸ = k) is δ i,j,k = min a b + c , ω . (2) (2c) Otherwise, for usual triangle, we count its angles; let they be α, β and γ, where α ⩾ β ⩾ γ. (2d) Then we set δ i,j,k = α -β γ . Let us especially note that δ, unlike σ, is calculated quickly, despite we need to consider ∼ n 3 triangles. Table 1 . The result of the recovery program using a greedy algorithm, which is some more complicated than the algorithm of [Melnikov and Trenina, 2018a] . In both cases, the branches and boundaries method is not used. Like [Melnikov and Trenina, 2018a] σ = 0.038, δ = 0.044 Let us also note the relationship of both of these criteria with the task we are considering: for example, for a "random" matrix, we obtained significantly worse results of calculation by the criterion δ, even for small dimensions; to say, for such a matrix of dimension 13 × 13 we obtained δ in limits about 0.4 -0.5, this is several times higher than the corresponding values for the "correct" matrices of dimension 28 × 28 and significantly lower percentage of initial fullness, see the next section.
Some results of computational experiments First of all, let us give a few comments on the given large tables of results. Both are given for the reader's possible verification of these results; at the same time, anyone can either simply recognize the table as a picture, or request from the authors, after which we shall send the same tables in the form of text. Having these tables, anyone can simply check their characteristics (badness, etc., according to the formulas given in the paper). It is possible to say, simplifying a little, that the topic of the article is how to obtain the missing values in these tables with minimum badness. As we have already noted, we evaluate the results of computational experiments well. Namely, an improvement in the performance of the algorithm was obtained (according to both criteria given in the previous section), compared with the simplest variant of the branch and boundary method, see [Melnikov and Trenina, 2018a; Melnikov and Trenina, 2018b] etc. More precisely, by the words "simplest variant", we mean that the simplest greedy heuristic used to select the next separating element. Here we apply a more complex greedy heuristic, while abandoning the method of branches and boundaries. It is clear that even more successful results (from the point of view of the quantitative criteria formulated above) we would have obtained by using both the branch and boundary method and a more complex greedy heuristic at the same time; however, it seems that we shall not satisfy acceptable time constraints. Though, we did not conduct detailed computational experiments for this case. To perform all the computational experiments described in the article, we used a computer with the following characteristics: Intel(R) Core(TM) i7-8700 CPU @ 3.20GHz In all our computational experiments, the total time of the computer was extremely short, and we did not record it, since it is significantly less than the time required to output the results of the algorithm (especially in comparison with the time necessary for the initial filling of only one cell of the matrix). For comparison, we repeat once again: such calculations by the method of branches and boundaries for dimensions of the order of 30 × 30 take about 1 second; the calculation of the distance by the Needleman -Wunsch algorithm between two sequences describing mtDNA takes about 3 minutes; and the filling of the entire matrix by the Needleman -Wunsch algorithm of the order of 30 takes about 1 day. Thus, let us briefly describe the computational experiments already carried out. In this paper, only one variant of the input data is used (28 monkeys, mt DNA, we briefly talked about this variant above); but we note in advance that similar results were obtained on all variants of the input data used. The initial matrix filled in as a result of the Needleman -Wunsch algorithm is given in [Melnikov and Trenina, 2018a, Tab. 8 ]. The initial matrix with about 10 % of remaining elements is given in [Melnikov and Trenina, 2018a, Tab. 9 ]. Let us remind once again that the calculations of the matrices in that paper were correct, but the calculations of the values σ and δ were erroneous. However, the mistakes did not affect the relative quality indicators of the algorithms. In the current paper, we present completely correct results, they are easy to check. The column designations in Table 3 are clear, and the row designations have the following meaning: (A) corresponds to the matrix, obtained by the best algorithm of [Melnikov and Trenina, 2018a] (that matrix was given on [Melnikov and Trenina, 2018a, Tab. 13] ); let us remind once again that the algorithm does not use branches and boundaries method; (B) corresponds to the matrix, obtained by the best algorithm of [Melnikov and Trenina, 2018b] (that matrix was given on [Melnikov and Trenina, 2018b, Tab. 17] ); the algorithm uses branches and boundaries method; (C) corresponds to the matrix, obtained by the simplest greedy algorithm of the current paper; the algorithm does not use branches and boundaries method; see its results (i.e., the obtained matrix and its characteristics) in The most successful values of σ and δ are highlighted in bold. A brief discussion of the obtained results is given in Conclusion, Section 7.
Some possible directions for further work on this subject Let us formulate some problems for the future solution, i.e., consider a brief description of the nearest directions for further work related to the modification and improvement of the described algorithms. For the first possible direction, we shall temporarily assume that the original processed algorithm under consideration (the Needleman -Wunsch algorithm is one of the possible examples only) is obviously not optimal, and requires improvement. Note that this fact, of course, is always true, regardless of everything else: for example, almost all the original algorithms (i.e., determining the distance between two given DNA sequences) are based on the long-known algorithm for constructing the Levenshtein metric (or Levenshtein distance), [Levenshtein, 1966] , to which the "penalties" are additionally added. At the same time, the numerical values of such penalties are always selected based on preliminary expert assessments (or even simply assumed to be equal to 1), [Christen, 2012a; Christen, 2012b; Yu et al., 2016; Suganthan et al., 2018] . However, it is clear that any selfstudy procedure should give an improvement of such values (penalties, etc.). Therefore, another inverse problem arises: to achieve such an improvement, where the minimization of the value δ is used as the criterion. In contrast to the first possible direction briefly described before, in other variants for further work we consider the given algorithm (the Needleman -Wunsch algorithm etc.) as something "God-given", i.e., not as the subject to change; we try, as above in this paper, to tune in to it. The second direction. We introduce a special "reliability coefficient" (let it be R < 1, to say, R = 0.9 in the following description), which we use as follows. We consider that the initial values of the matrix (in the example considered in the paper, the remaining 10 % of the elements after the removal) have a weight of 1.0. The elements derived from only the initial ones (i.e., in the beginning of filling) have a weight of R. And in the general case (i.e., after filling in some elements) we proceed as follows. As in the greedy algorithm already discussed in this paper, we form all possible triangles obtained together with the element selected for filling, i.e. if the considered unfilled element of the matrix is m i,j , then, as before, we consider all such k that m i,k and m j,k are already filled. However, we calculate the obtained values with the reliability coefficients already assigned to these values, i.e., we minimize the general function, which includes values with these coefficients; for the reliability coefficients R i,k and R j,k , we assume that the reliability coefficient of the considered triangle is R ∆ = R i,k + R j,k 2 . (3) The resulting value obtained as a result of minimization is placed in a matrix with its new reliability coefficient equal to the a priori value of R multiplied by the average reliability coefficient of all considered triangles that form the element m i,j : using (3) and assuming we are considering m triangles, this new coefficient can be written as follows:
R (1) ∆ + R (2) ∆ + + R (m) ∆ R m . And, of course, the best value of the reliability coefficient R should be obtained as a result of some selflearning process. The third direction. Here we propose to continue the simultaneous application of increasingly complex greedy heuristics and the method of branches and boundaries. We hope that the results will surpass those obtained in this paper and in [Melnikov and Trenina, 2018a; Melnikov and Trenina, 2018b] . At the same time, we note that almost no time is spent on the method of branches and boundaries, at least in comparison with the time that needs to be spent on filling in only one initial element of the matrix. The fourth direction. This is a detailed study of the quality of algorithms depending on the dimension of the matrix and the percentage of its initial fullness. (Note that we have not actually started solving this problem yet.) The fifth direction. We believe that it is possible to choose which elements of the matrix should be initially filled, of course, within a predetermined total number of them. This possibility sometimes reflects the subject area under consideration: after all, the total time of such filling will practically not depend on specific elements, but will depend on their number only. Is it true that in this case, the elements for the initial filling should be selected so that there would be approximately the same number of them in all rows of the matrix? (Answering this question is the fifth possible direction of work.) The sixth direction represents a new approach to comparing different heuristics for distances between DNA chains, i.e., an alternative approach to the one discussed in [Melnikov, Pivneva, and Trifonov, 2017] and some of our other papers cited there. Namely, after filling in several distance matrices with various algorithms (i.e., algorithms for obtaining distances between pairs of DNA chains), we consider all possible triangles in these matrices; note again that their number is quite large, of the order ∼ n 3 . Next, for each initial filling algorithm, we consider a list of these triangles, ordered, for example, by non-increasing values of the badness (considered, for example, as δ i,j,k , see ( 2 )). The main idea of this heuristics is that we assume that all the algorithms described in the literature and on the Internet for obtaining distances between pairs of DNA chains are logically correct. Therefore, considering some "natural" metric on such ordered sequences of triangles, for the "best" algorithm for the initial filling of matrices (the best algorithm for calculating the distance between pairs of DNA chains), we obtain the minimum value of the sum of the distances to other ordered sequences of triangles. As such a natural metric on ordered sequences of triangles, we can choose some natural function from the pairwise correlation between the sequences. In our preliminary calculations, we choose a linear function as such one: having a value 0 in the case of matching sequences; having a value 1 (the maximum possible value) in the case of the maximum possible number of the minimum number of exchanges required to convert from the first sequence to the second one; note that for a matrix of the order n × n, the number of its triangles is ∼ n 3 , therefore the number of possible exchanges is ∼ n 6 ; and intermediate values otherwise; these values are calculated, as we already noted, using the simplest linear function. Note that our version of the pairwise correlation is obtained here with another version of the linear function, i.e. when simultaneously replacing 0 with 1 and 1 with -1 in the items above. After that, we propose to consider "pairwise correlation between pairwise correlations": for this, we need to arrange the heuristic algorithms for the initial placement of DNA chains in two ways (i.e. according to [Melnikov, Pivneva, and Trifonov, 2017] and according to the above). The seventh direction. And of course, as a possible direction for further work, it is necessary to consider new objects of application of the described algorithms: other species (besides monkeys), Y-chromosomes instead of mt DNA s, other initial filling algorithms (instead of Needleman -Wunsch) . . . Besides, for monkeys, we propose to consider a very strong increase in dimension (it is optimal to consider all types, 500 +) with a simultaneous decrease in the percentage of initially filled matrix cells (to say, to 5 % instead of 10 %). Of course, these seven directions do not limit further possible work on the topics described here . . .
Conclusion In this article, we propose an improved algorithm for recovering missing data in distance matrices. An unusual feature of the results is that the reduction of the quality criteria σ and δ used in several of our articles by different algorithms occurs independently of each other, i.e., there is a binary relationship between the algorithms, which consist in the fact that the first of them gives the best results for both criteria, forms a partial order only. Similar relative results are obtained for other objects of study (not for monkeys etc.). Of course, the best option would be the simultaneous minimization of σ and δ, which we shall achieve in the subsequent work. However, the results presented in this paper are of big interest.
Acknowledgement This work was supported by the Natural Science Foundation of Guangdong Province (No. 2019A1515110971) and Shenzhen National Science Foundation (No. 20200827173701001). the "badness" of corresponding triangle; it is usually counted in the following way.(2a) Firstly, we rename m i,j , m i,k and m j,k into a, b and c, where a ⩾ b ⩾ c.
Table 2 . 2 , the value of the table 315 corresponds to the obtained value 0.315, etc. The result of the recovery program using a complicated greedy algorithm. The branches and boundaries method is not used. 0 313 258 334 328 341 334 326 505 324 344 421 315 334 335 335 262 334 332 337 334 997 506 329 338 325 325 344 313 0 330 313 303 342 323 222 505 320 340 420 312 313 334 334 297 328 331 332 292 997 504 269 336 316 320 342 258 330 0 335 343 339 333 320 505 327 347 422 319 335 334 334 296 332 331 343 341 997 505 335 337 273 309 344 334 313 335 0 329 340 292 330 505 327 344 420 328 311 334 334 332 322 333 333 257 997 503 333 335 333 330 344 328 303 343 329 0 343 332 236 505 330 341 417 328 329 336 336 325 333 334 304 327 997 505 329 338 320 328 343 341 342 339 340 343 0 341 341 505 341 351 421 341 340 341 341 341 341 341 344 337 997 506 343 341 341 341 345 334 323 333 292 332 341 0 332 505 318 344 421 326 329 335 335 334 279 334 337 331 997 499 334 331 334 332 344 326 222 320 330 236 341 332 0 505 329 344 420 326 330 336 336 322 333 333 333 330 996 504 325 337 298 324 344 505 505 505 505 505 505 505 505 0 505 505 505 506 505 505 505 505 505 505 505 505 996 523 505 505 505 505 505 324 320 327 327 330 341 318 329 505 0 344 421 302 348 354 354 328 350 351 337 333 996 508 333 354 332 347 360 344 340 347 344 341 351 344 344 505 344 0 419 344 359 361 361 344 361 360 326 341 996 509 332 360 344 359 268 421 420 422 420 417 421 421 420 505 421 419 0 420 427 428 428 420 428 427 315 419 996 514 420 427 420 427 428 315 312 319 328 328 341 326 326 506 302 344 420 0 355 361 361 297 358 357 337 332 996 509 332 360 330 353 366 334 313 335 311 329 340 329 330 505 348 359 427 355 0 361 361 355 358 359 356 264 999 678 355 361 356 357 367 335 334 334 334 336 341 335 336 505 354 361 428 361 361 0 301 358 357 264 362 361 997 534 401 300 401 364 367 335 334 334 334 336 341 335 336 505 354 361 428 361 361 301 0 358 355 321 362 361 997 534 401 256 401 364 367 262 297 296 332 325 341 334 322 505 328 344 420 297 355 358 358 0 359 326 337 331 996 509 330 342 327 354 366 334 328 332 322 333 341 279 333 505 350 361 428 358 358 357 355 359 0 356 361 359 996 287 360 265 359 359 367 332 331 331 333 334 341 334 333 505 351 360 427 357 359 264 321 326 356 0 360 358 997 534 399 310 398 361 367 337 332 343 333 304 344 337 333 505 337 326 315 337 356 362 362 337 361 360 0 303 996 509 336 361 336 359 365 334 292 341 257 327 337 331 330 505 333 341 419 332 264 361 361 331 359 358 303 0 997 509 330 360 333 357 366 997 997 997 997 997 997 997 996 996 996 996 996 996 999 997 997 996 996 997 996 997 0 995 997 997 997 997 997 506 504 505 503 505 506 499 504 523 508 509 514 509 678 534 534 509 287 534 509 509 995 0 509 531 509 533 535 329 269 335 333 329 343 334 325 505 333 332 420 332 355 401 401 330 360 399 336 330 997 509 0 401 332 395 404 338 336 337 335 338 341 331 337 505 354 360 427 360 361 300 256 342 265 310 361 360 997 531 401 0 401 397 401 325 316 273 333 320 341 334 298 505 332 344 420 330 356 401 401 327 359 398 336 333 997 509 332 401 0 244 405 325 320 309 330 328 341 332 324 505 347 359 427 353 357 364 364 354 359 361 359 357 997 533 395 397 244 0 402 344 342 344 344 343 345 344 344 505 360 268 428 366 367 367 367 366 367 367 365 366 997 535 404 401 405 402 0 σ = 0.079, δ = 0.103 0 313 258 332 328 341 332 326 505 317 344 317 286 332 330 330 262 332 329 333 332 997 332 329 332 270 270 344 313 0 330 284 245 342 284 222 505 315 340 316 311 284 317 285 297 280 321 302 292 997 273 269 286 316 315 342 258 330 0 334 343 339 332 320 505 320 347 340 283 334 330 330 269 332 329 343 341 997 332 335 331 273 264 344 332 284 334 0 285 340 292 283 505 325 344 329 325 253 322 285 328 282 325 293 257 997 276 283 285 327 327 344 328 245 343 285 0 343 284 236 505 326 341 323 325 280 323 282 322 277 326 304 295 997 273 267 281 320 326 343 341 342 339 340 343 0 341 341 505 341 351 332 341 340 341 341 341 341 341 344 337 997 341 343 341 341 341 345 332 284 332 292 284 341 0 282 505 318 344 318 322 280 324 283 327 279 327 292 287 997 276 281 281 327 329 344 326 222 320 283 236 341 282 0 505 324 344 314 324 276 324 278 321 276 326 297 290 996 271 262 277 298 323 344 505 505 505 505 505 505 505 505 0 505 505 505 506 505 505 505 505 505 505 505 505 996 505 505 505 505 505 505 317 315 320 325 326 341 318 324 505 0 344 313 302 314 309 307 324 315 311 330 329 996 310 329 311 325 312 327 344 340 347 344 341 351 344 344 505 344 0 325 344 326 360 360 344 328 359 326 341 996 355 332 356 344 359 268 317 316 340 329 323 332 318 314 505 313 325 0 311 308 310 307 310 311 311 315 309 996 308 306 310 306 311 323 286 311 283 325 325 341 322 324 506 302 344 311 0 309 306 305 297 310 308 330 330 996 306 329 308 291 276 354 332 284 334 253 280 340 280 276 505 314 326 308 309 0 304 266 337 268 308 300 264 999 282 292 268 336 309 353 330 317 330 322 323 341 324 324 505 309 360 310 306 304 0 301 308 303 264 304 301 997 300 299 300 331 307 355 330 285 330 285 282 341 283 278 505 307 360 307 305 266 301 0 335 264 321 297 291 997 289 288 256 330 335 355 262 297 269 328 322 341 327 321 505 324 344 310 297 337 308 335 0 338 326 330 329 996 338 328 332 264 272 354 332 280 332 282 277 341 279 276 505 315 328 311 310 268 303 264 338 0 306 300 294 996 287 291 265 336 336 353 329 321 329 325 326 341 327 326 505 311 359 311 308 308 264 321 326 306 0 307 305 997 304 303 310 334 305 355 333 302 343 293 304 344 292 297 505 330 326 315 330 300 304 297 330 300 307 0 303 996 300 293 300 327 336 353 332 292 341 257 295 337 287 290 505 329 341 309 330 264 301 291 329 294 305 303 0 997 294 288 293 327 336 354 997 997 997 997 997 997 997 996 996 996 996 996 996 999 997 997 996 996 997 996 997 0 995 997 997 997 997 997 332 273 332 276 273 341 276 271 505 310 355 308 306 282 300 289 338 287 304 300 294 995 0 265 294 336 330 349 329 269 335 283 267 343 281 262 505 329 332 306 329 292 299 288 328 291 303 293 288 997 265 0 294 327 330 347 332 286 331 285 281 341 281 277 505 311 356 310 308 268 300 256 332 265 310 300 293 997 294 294 0 334 328 346 270 316 273 327 320 341 327 298 505 325 344 306 291 336 331 330 264 336 334 327 327 997 336 327 334 0 244 344 270 315 264 327 326 341 329 323 505 312 359 311 276 309 307 335 272 336 305 336 336 997 330 330 328 244 0 344 344 342 344 344 343 345 344 344 505 327 268 323 354 353 355 355 354 353 355 353 354 997 349 347 346 344 344 0
Table 1 1 of this paper; (D) corresponds to the matrix, obtained by the compli- cated greedy algorithm of the current paper; the algorithm does not use branches and boundaries method; see its results in Table 2 of this paper; Certainly, all the calculation results shown in this table can be quickly checked using a simple supportive com- puter program.
Table 3 . 3 General results of some computational experiments T σ δ (A) 0.091 0.110 (B) 0.029 0.133 (C) 0.079 0.103 (D) 0.038 0.044
I.e., when considering summation, as well as when taking minima and maxima.
In some our previous papers, we wrote that the number of such violations ranged from 2 (the minimum value in previous calculations) to several dozen (for matrices
about 30 × 30); it depended on the subject area, as well as on the specific algorithm. In the case under consideration here (28 species of monkeys of different genera, mt DNA, Needleman -Wunsch algorithm), we obtained no such violations at all, then the item (2b) was not used.
|
10.5888/pcd15.180026
|
cc-by|public-domain
| null | null |
openalex
|
Background A growing body of literature shows that health promotion and disease prevention strategies and messages may not be effective in reaching racially and ethnically diverse communities, unless those strategies are culturally and linguistically adapted for target communities (1) . The Racial and Ethnic Approaches to Community Health for Asian Americans (REACH FAR) project is a multilevel, evidence-based program of health promotion and disease prevention for Asian American communities in New York and New Jersey. Guided by a socio-ecological framework, social marketing principles, and a community based participatory approach, the project implemented multilevel, evidence-based strategies culturally adapted to address hypertension and improve access to healthy food options for Asian Americans in various community settings (1) . The strategies were delivered through a multisector coalition made up of a lead academic agency, New York University Center for the Study of Asian American Health; the NYC Department of Health and Mental Hygiene (NYC DOHMH); 4 community-based organizations: UNITED SIKHS, serving the Asian Indian population; Diabetes Research, Education, and Action for Minorities, serving the Bangladeshi community; Kalusugan Coalition, Inc., serving the Filipino community; Korean Community Services of Metropolitan New York, Inc., serving the Korean community; and other organizations and groups serving these communities. Coalition partners implemented strategies at various community sites, including faith-based organizations, ethnic restaurants, grocery stores, pharmacies, and primary care practices. REACH FAR has 2 program arms to deliver culturally and linguistically adapted resources. The first arm is focused on improving access to healthy foods and beverages and includes implementation of policies adapted for communal meals at faith-based organizations, healthy food options and labeling at restaurants, and strategic placement and discounting of healthy food products at grocery stores (2) . The second arm is focused on improving access to hypertension management and cardiovascular disease prevention programs by offering the NYC DOHMH's Keep on Track (KOT) blood-pressure monitoring program at faith-based organizations and increasing access to Million Hearts (https://millionhearts.hhs.gov/) blood pressure medication adherence resources at faith-based sites, community-based pharmacies, and health care providers' offices (3). The REACH FAR coalition leveraged existing partnerships and fostered new relationships with implementation sites to tailor and disseminate strategies for their respective communities. Geographic areas with high concentrations of targeted Asian American communities were selected at project conception. Hence, a primary motivation for creating our map was to assess the population and geographic reach of the REACH FAR project by comparing Asian American population clusters (ie, geographic areas with high concentrations of Asian Indian, Bangladeshi, Filipino, and Korean populations) with the locations of implementation sites and participants. A desire lines (or spider diagram) approach was used to visualize KOT program participation. Mapping provides a visual means of evaluating program reach and is a concise way of communicating the role of implementation sites in delivering culturally, linguistically, and geographically targeted health prevention strategies.
Methods We geocoded the addresses of 12 implementation sites active as of 2016 and residential addresses of KOT program participants reported in baseline surveys from 2015 and 2016 (4-6). All research conducted with human participants was reviewed by the NYU School of Medicine Institutional Review Board and approved as an expedited study. Data processing was performed with version R version 3.4.1 (R Foundation for Statistical Computing). Straight lines were created between successfully geocoded participants' residences (n = 587; 86% of participants) and faith-based organizations (n = 12); each line represents a KOT program participant (7) . Population estimates from Census 2010 Summary File 1 data and household language from ACS 2015 5-Year estimates were obtained for Census tracts in NY and NJ (8, 9) . Processed data were exported as Esri shape files (10) . The map construction and geo-processing related to population clusters were completed with ArcGIS 10.4.1 (Environmental Systems Research Institute [Esri]). The Asian American subgroup population clusters are significant hot spots, positive z scores, determined by the ArcGIS Hot Spot Analysis (Getis-Ord Gi*) tool (Esri) with the Contiguity Edges Corner and Apply False Discovery Rate correction parameters; the shading indicates intensity of clustering with darker shading indicating more clustering of high values (larger z scores). Overlaps in population clusters were found by using the ArcMap Intersect tool, version 10.3 (Esri).
Main Findings An overarching objective of REACH FAR is delivery of targeted strategies for 4 Asian American communities. The map demonstrates that program implementation sites are located in population clusters of targeted Asian American subgroup populations, as intended at program inception: Asian Indian in Middlesex and Queens counties, Bangladeshi in Kings and Queens counties, Filipino in Queens county, and Korean in Queens and Bergen counties. By mapping KOT program participants, we found that large concentrations of congregants residing in areas neighboring each faith-based site are reached by the KOT program. However, the maps also demonstrate that the programs also reached community members residing outside of immediate or neighboring ethnic enclaves. We suspect that people attending these events prefer or need culturally and linguistically adapted resources that are unavailable in the neighborhoods in which they reside. Culturally and linguistically adapted materials regarding healthy eating and blood pressure control developed for limited English-proficient Asian Americans were disseminated at implementation sites (11), reaching 1,353,201 people as of September 30, 2017. The map reveals opportunities for collaboration, the areas where population clusters overlap, and gaps in coverage in areas with population clusters where the project did not have implementation sites.
PREVENTING CHRONIC DISEASE
Action Our map provides a visual illustration of the network of faithbased organizations and community-based organizations coordinating to promote healthy eating and heart health and the progress of implementation as illustrated with the KOT program. To this end, mapping products are being used in the following ways to enhance future coordination and collaboration between partners: The map is being presented to the NYC DOHMH to demonstrate the need for reaching Asian American populations. Before our coalition efforts, the KOT program had not been implemented in any faith-based organizations serving Asian Americans. Our results demonstrate both success and potential for future NYC DOHMH engagement efforts with Asian American communities.
1. The map is being reviewed at coalition meetings to discuss opportunities for scaling the program to expand program reach. For example, the map suggests that partnering with community-based organizations serving the Asian Indian and Filipino communities in Hudson County or the Bangladeshi community in Bronx County may expand reach. 2. opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions.
Centers for Disease Control and Prevention www.cdc.gov/pcd/issues/2018/18_0026.htm
|
10.7759/cureus.8736
|
cc-by
| null | null |
openalex
|
Background Shared governance is considered a model for mounting autonomous decision making in nursing profession and practice. This study aimed to assess how registered nurses in an outpatient department in a tertiary care hospital perceive shared governance.
Methods We conducted a cross-sectional study among a convenient sample of registered nurses in an outpatient department. A self-administered, Index of Professional Nursing Governance (IPNG) questionnaire was used to measure the study outcome. A descriptive analysis was used to describe nurses' characteristics and study outcomes.
Results A total of 186 nurses completed the questionnaire. Of whom, 151 (92.1%) were female, and 78 (47.3%) were aged between 20 and 30 years. Only 54 (29.3%) and 59 (31.7%) had indicated a shared decision in terms of controls and influence scales, respectively. The majority of the nurses indicated traditional shared across shared governance scales except in the access information scale.
Conclusion The findings showed a prevalent traditional nursing management style in the study setting. Supportive strategies and education must be provided for both managers and staff nurses to develop and implement shared governance in their practice.Introduction In a complex health care system, nurses are a vital component in providing optimal patient quality care and promote outcomes. Nursing nowadays facing multifaceted challenges, like shortages in the workforce, increased workloads, and patient acuities that have reshaped the attention to the quality of nursing care while fulfilling increased patient needs and demands. In addition to these challenges, nurses are astounded with increased rules that inflate their work profile and load, increase job dissatisfaction, and decrease bedside time spent with the patient. Although these challenges increased nurses' responsibility and accountability to accomplish professional practice, it was not escorted with increasing power or authority to figure out required changes to impact nursing practice . Therefore, healthcare organizations have developed several professional practice models to direct disciplines' clinical practice, empowering and authorizing health staff, and improve provided quality of care [2, 3] . The nursing profession has encouraged and supported nursing involvement in nursing practice models such as shared governance . Nurse participation in shared governance guarantees accountability for the safety and quality of care and the autonomy of the nurses . Shared governance has been defined as "a decentralized approach which gives nurses a greater authority and control over their practice and work environment; engenders a sense of responsibility and accountability; and allow active participation in the decision-making process, particularly in the administrative area from which they were excluded previously" . Governance generally comprises structure and process where a group of individuals direct, control, and regulate their goal-oriented efforts . Shared governance as an official program should include nurses in control and authority decisions by attaining the right to control their practice and extending their governance beyond that to higher and different tasks like scheduling, and evaluating personnel, budgeting, that were historically controlled solely by managers . Moreover, shared governance is a model that includes shared decision-making between the healthcare workforce members and is centered on the principles of partnership, equity, accountability, and ownership [7, 8] . Shared governance structure supports patient care directly, promotes nurses' control over their practice and accountability for quality patient care . For nurses, it results in feelings of empowerment that allow for professional autonomy. The application of shared governance structure leads to enhance the provision of quality of care [4, 9] , cultivate collaboration between healthcare professionals, advance the quality of care and clinical effectiveness; upsurge employees confidence; assist in developing individual and professional skills; growth of professional profile; which in turn lead to improve personal communication; advance knowledge and skills; intensification of professionalism and accountability; reduce duplication of effort [4, 10, 11] . Nurses' satisfaction is a vital end-point of shared governance. Investigating shared governance among nurses at King Fahad Medical City is important because of the influence of attention on human factors in nursing and maintain workforce retention. Therefore, this study aimed to assess the perception of shared governance among nurses in a tertiary care hospital, Riyadh, Saudi Arabia.
Materials And Methods
Study design and settings A cross-sectional study was conducted to assess perceptions of shared governance levels among nurses. A convenient sampling method was used for recruiting the nurses working in ambulatory care in a tertiary care hospital, Riyadh, Saudi Arabia. All ambulatory care nurses were invited to participate in the study by an invitation letter attached to a copy of the questionnaire and cover sheet describing the study's objective and voluntary participation. Participants' identity was kept anonymous.
Sample characteristics A total of 186 nurses included in the study. The majority of participating nurses 151 (92.1%) were females, and 148 (92.8%) were staff nurses. About 78 (47.3%) of the nurses were aged between 20 and 30 years. One-hundred and thirty (79.9%) had a bachelor degree, while 25 (15.2%) had a nursing diploma. Nearly half of the nurses had six-ten years of experience, and 119 (72.5%) had one-five years of experience in the current institution (Table 1 ).
TABLE 1: Participants' demographics Table 2 displays the descriptive statistics for the six scales of the IPNG scale. Regarding the "Control" scale, the results showed that slightly more than two-thirds (68.5%) of the nurses (n=126) perceived traditional decision making and 54 (29.3%) had indicated a shared decision making. Furthermore, 122 (65.6%) of the nurses perceived traditional decision making in the "Influence" scale compared to 59 (31.7%) who perceived a shared decision making. The results of "Participation" scale indicated that more than half of the nurses (54.1%) perceived having a traditional decision making. Moreover, the "Ability" scale showed a traditional decision making among 110 (59.5%) of the nurses. Nevertheless, 72 (39.9%) of the nurses revealed the "Ability" scale is governed by the shared decision making. (
Discussion Overall, the study showed a traditional management decision making as indicated by the nurses. The distributions of the percentages of nurses' responses were all more or less asymmetrical and concentrated to the left side of the chart, indicating a traditional model of governance. The most proportioned distribution that makes relatively somewhat balance between nurses' shared governance model, and the traditional model of governance was for the participation scale. Overall, the findings reflect the administrative driven model of governance at the study site, which means that the governance and management type had no effect on the perception of staff nurses and that nurses managers are working in an environment that is not equally sharing decisions and not enabling staff nurses to control over their practice. In the control scale, the results indicated that nurses had limited control over their practice. The findings were inconsistence with the similar studies reported in the literature which revealed that nurses' perceptions of their work setting are more related to the shared governance model [12, 13] . These studies indicated that nurses and administration were equally involved in decision-making activities concerning their control over professional practice [12, 13] . Relating to the influence over practice and resources, participating nurses perceived they lack influence or formal authority in several daily procedures, including patient care locations, obtaining and monitoring supplies, patients' admissions, and discharges, creating new clinical and administrative positions, and generating schedules. The results disagree with Hashish et al., who reported that nurses indicated high influence over practice and resources . Along with the findings of the current study align with Seada and Etway who showed that nurses perceived various parts of their influence over different activities are being done only with an administrator decision with limited staff inputs . The findings of our study showed that nurses perceived a lack of shared ability with nursing management to engage in nursing profession committees mostly concerning their clinical practices, staff scheduling, and strategic planning. Moreover, nurses perceived they have limited ability to play a part in committees that relating multidisciplinary professionalism, organizational expenses, and budgets. George et al. revealed that nurses from a non-shared governance hospital had less engagement in decision making compared to nurses in a shared governance model, which comes in line with our results . However, several studies have reported results supporting nurses' ability to participates in nursing profession committees [12, 13] . The findings of this study were consistent with results of a study conducted by Tourangeau et al. ; they stated that nurses perceived they have the least extent of control over professional practice; as well as, they perceived slight contribution or control in several areas that affect directly the patients' bedside care from nursing, quality care standards, educational progress, and determining the structure of nursing care for their work. Also, Seada and Etway revealed that nurses had lowermost scores regarding their perception of shared governance which showed that they did not have control over their professional work setting . Shared governance is characterized by collaborative decision-making between management and staff working, together at the organizational level and unit level . The values of shared governance have been united in nursing structures to providing a transformational structure for staff nursing care and enhancing an organization's productivity and performance . The three essential values related to shared governance are the responsibility of delivering for nursing care should belong to clinical staff, nurses' authority for being renowned by the organization, and quality patient care accountability and nursing professionalism must be acknowledged by the clinical staff . Nursing shared governance denotes to the played nurses' role in decision-making and liability for patient care . Shared governance affords the context for a cooperative milieu of nursing leaders and nurses. Both, they can frame a partnership of shared decision for operational and clinical practices . Worldwide, nurses are the prime profession in healthcare settings. Therefore, participation and influence in shared governance are necessary to enable constant transformation, advance and progress of the nursing profession. Bringing this to reality, fitting models, and processes that deliver and inspire participation in decision-making are indispensable .
Limitations Limitations of this study include the employment of basic descriptive statistics. A further bivariate and multivariate analysis could be considered in the future. Other acknowledged limitations are the relatively modest sample size and limited study site; therefore, the findings may not be representative of the entire. A prospective study with a bigger sample size and multi-settings will be powerful to generalize the data.
Conclusions The results showed that the studied setting lacks a shared governance model in place to engage nurses in decision-making which can enable them to control their professional practice. The findings of our study showed a traditional management governance type in the study setting. The study findings can be utilized to enhance nurses' work milieu and improve shared governance. These results would be an added value for nursing top management to enhance nurses' perception of shared governance by emerging or implementing shared governance model. In the light of our findings, nurse managers must adopt and apply strategies to empower nurses like shared governance that provide nurses the chance to control their nursing practice and improve quality nursing care. Table 2 ) 2 Factor Traditional (management) decision Shared decision making Nurses are the decision maker scales making (86-172) n(%) (173-344) n(%) (345-430) n(%) Controls 126 (68.5) 54 (29.3) 4 (2.2) Influence 122 (65.6) 59 (31.7) 5 (2.7) Official authority 124 (67) 56 (30.3) 5 (2.7) Participation 100 (54.1) 81 (43.8) 4 (2.1) Access information 74 (40.2) 105 (57.1) 5 (2.7) Ability 110 (59.5) 72 (39.9) 3 (1.6)
TABLE 2 : Sub-scales of nursing shared governance 2
Kaddourah et al. Cureus 12(6): e8736. DOI 10.7759/cureus.8736
|
10.5194/isprs-annals-v-3-2022-131-2022
|
cc-by
| null | null |
openalex
|
Calculating solar-sensor zenith and azimuth angles for hyperspectral images collected by UAVs are important in terms of conducting bi-directional reflectance function (BRDF) correction or radiative transfer modeling-based applications in remote sensing. These applications are even more necessary to perform high-throughput phenotyping and precision agriculture tasks. This study demonstrates an automated Python framework that can calculate the solar-sensor zenith and azimuth angles for a push-broom hyperspectral camera equipped in a UAV. First, the hyperspectral images were radiometrically and geometrically corrected. Second, the high-precision Global Navigation Satellite System (GNSS) and Inertial Measurement Unit (IMU) data for the flight path was extracted and corresponding UAV points for each pixel were identified. Finally, the angles were calculated using spherical trigonometry and linear algebra. The results show that the solar zenith angle (SZA) and solar azimuth angle (SAA) calculated by our method provided higher precision angular values compared to other available tools. The viewing zenith angle (VZA) was lower near the flight path and higher near the edge of the images. The viewing azimuth angle (VAA) pattern showed higher values to the left and lower values to the right side of the flight line. The methods described in this study is easily reproducible to other study areas and applications.INTRODUCTION Remote sensing has proved to be highly effective and efficient in studying a diverse variety of natural and ecological resources. Other than satellite and aerial remote sensing, recent advances in Unmanned Aerial Vehicles (UAV) and sensor technology has opened more opportunities to study vegetation dynamics, specifically in agricultural applications (Maddikunta et al., 2021) . Since UAVs can be flown at lower altitudes than satellites or aircrafts, the resulting products offer higher spatial resolution and with more accurate canopy spectra (Tao et al., 2020) . The canopy spectra can be used to model or represent different plant traits. For instance, different vegetation indices (e.g., normalized difference vegetation index, NDVI) can indicate overall crop health that improves precision agriculture practices (Radoglou-Grammatikis et al., 2020) . Additionally, UAV sensors offer highthroughput plant phenotyping that accelerates current crop breeding operations (Song et al., 2021) . Moreover, UAV-based imageries can be used to train advanced machine learning models, which predict various crop traits, disease, yield, and seed quality at plot-level (Bhadra et al., 2020; Maimaitijiang et al., 2020; Nguyen et al., 2021) . Hyperspectral sensors can collect reflected spectra from crop canopies with higher spectral resolution. A typical hyperspectral image (HSI) often contains hundreds or even thousands of bands for a wide range of wavelengths. Generally, the wavelengths can vary from Very Near Infrared (VNIR, 400-1000 nm) to Shortwave Infrared (SWIR, 900-2500 nm) with different spectral resolution (1 to 10 nm). Plants reflect electromagnetic radiation, which contains information about their biophysical composition and physiological status (Segarra et al., 2020) . Numerous studies * Corresponding author have utilized the broader range of HSI products to study different characteristics of plants and vegetation (Mariotto et al., 2013; Fernandes et al., 2015; Banerjee et al., 2020; Wang et al., 2021) . The quality of HSI-based inference heavily depends on the accuracy of HSI post-processing techniques. Generally, the HSI sensor provides the raw Digital Number (DN) or radiance (in Wsr -2 m -2 ), which is then converted to unitless top-ofatmosphere (TOA) reflectance and surface reflectance (SR). Empirical Line Method (ELM) is the widely used calibration technique to convert radiance into SR using different calibration targets on the ground (Markelin et al., 2008; Wang and Myint 2015; Ortiz et al., 2017) . The principal assumption behind this technique is that the objects on the ground represent a Lambertian surface, which appears uniformly bright from all directions of view and reflects the entire incident light (Mao et al., 2020) . However, the crop canopy architecture is far from being a Lambertian surface and exhibits anisotropic effects (Jiao et al., 2014) . Therefore, several studies have identified that multiple viewing angles or viewing geometry of sensors play an important role in the pixel-level SR (Vermote et al., 2009; Zhang et al., 2014) . For example, Galvao et al., (2009) retrieved highly accurate Vegetation Indices (VIs) from Hyperion and MODIS satellite data when using backward observations. Similarly, Gu et al., (2015) found improved Leaf Area Index (LAI) estimation accuracy from backward observations compared to forward observations in CHRIS/PROBA data. Huang et al., (2011) demonstrated that multi-angular hyperspectral observations could retrieve the vertical distribution of chlorophyll content in winter wheat. In terms of UAV-based observations, several studies have utilized snapshot (or frame) hyperspectral cameras to derive multi-angular spectral information. For instance, Roosjen et al., (2018) achieved improved results in estimating LAI and chlorophyll content of potato by using multi-angular data. They introduced a goniometer-based simulation method for HSI footprints with high overlaps. The multiple viewing angles were converted to zenith and azimuth angles, which were used to simulate PROSAIL spectra and derive better LAI and chlorophyll retrieval accuracy. Similarly, Mao et al., (2020) found that the effect of multi-angular observations was significant in deriving VIs for soybean and maize. They also extrapolated different viewing angles from a snapshot hyperspectral camera mounted with a UAV and corrected for the Bi-directional Reflectance Function (BRDF) effect. Therefore, the availability of multiple solar-sensor zenith and azimuth angles is highly important to accurately study different plan characteristics. Alternative to snapshot cameras, push-broom hyperspectral sensors (or line-scanner sensors) are now widely used with UAVs. The push-broom sensor captures one line per exposure that forms one image line after the other (Barreto et al., 2019) . Therefore, push-broom sensors can outperform snapshot cameras, as the latter systems require a compromise between spatial coverage, spatial resolution, and spectral resolution (Aasen et al., 2015; Yi et al., 2021) . However, extracting the solar-sensor zenith and azimuth angles from a push-broom sensor is not as straightforward as snapshot cameras. While the snapshot camera provides 2D scenes captured across overlapping flight lines in relatively higher time interval, the push-broom sensor captures line by line 1D spectra across its flight path. Due to the line-by-line scanning mechanism, push-broom sensors suffer from wind-related motions during data acquisition (Jaud et al., 2018) . As a result, push-broom sensors require high accuracy Global Navigation Satellite System (GNSS) and Inertial Measurement Unit (IMU) onboard the UAV to ortho-rectify the lines and generate a geometrically accurate hyperspectral cube (Yuan and Zhang 2008) . Due to the availability of GNSS/IMU system onboard the platform, the solar-sensor geometry can be directly calculated using linear algebra and spherical trigonometry. Therefore, the objective of this study is to develop an automated framework that can calculate the solar-sensor zenith and azimuth angles for each pixel in a hyperspectral cube collected by a push-broom UAV scanner with cross-grid flight pattern.
STUDY AREA AND DATASETS
Experimental Setup The experiment was setup in the Planthaven Farms at OFallon, Missouri, United States (Figure 1 ). The site was located slightly northeast from Saint Louis city close to the Mississippi River to the north. The field was planted with 220 rows of maize on May 25, 2021, where 2 rows were marked as one plot. Total 55 different genotypes or cultivars of maize were planted with 2 replicas. The field was approximately 75 m long and 20 m wide. During the growing season, average temperature was between 23-24C and average annual precipitation was 1092.2 mm for the study area.
UAV Flight A DJI M600 Pro UAV was used to collect the hyperspectral data for the study area (Figure 1c ). The UAV was equipped with a Headwall Nano-Hyperspec VNIR push broom camera (Headwall Photonics, Massachusetts, United States), a FLIR Vue Pro thermal camera (FLIR Systems, Oregon, United States), and an APX-15 GNSS/IMU (Applanix Corporation, Ontario, Canada) unit all attached to a DJI Gimbal (Figure 1d ). The APX-15 UAV GNSS/IMU records the precise time, position, and orientation of the sensor at 200 Hz interval. Full specifications of the sensor and the GNSS/IMU unit is provided in Table 1 . Two UAV flights were conducted on July 20th and August 4th of 2021. Each flight was planned in a cross-grid pattern (Figure 1f ) in UgCS mission planning software (v4.0.187, SPH Engineering, Latvia) with 4 length wise and 9 width wise lines, resulting in total 13 hyperspectral cubes. The altitude and velocity for both flights were 50 m and 3 m/s. The ground sampling distance (GSD) was found 3.01 cm from both flights.
METHODS The extraction of solar-sensor geomtery contain three major parts (Figure 2 ): 1) hyperspectral cube processing, 2) locating viewing point for each pixel, and 3) calculating solar-sensor geometry. The process was automated using Python libraries which are available in a public repository with test datasets (https://github.com/remotesensinglab/uav-solar-sensor-angle).
Locating Viewing Point for Each Pixel The viewing point in terms of each pixel was required to calculate both sensor zenith and azimuth angles (Figure 2b ). First, the GNSS data was extracted from APX-15 device and converted to an ASCII text file which contained latitude, longitude, and timestamp information. Additionally, the coordinates of each pixel was calculated by converting the raster data into a geospatial text file using GDAL v3.3.1 (GDAL/OGR 2020). The raster image also had longitude, latitude, and timestamp information. Therefore, only the corresponding GNSS observations for a HSI cube were filtered by matching the timestamp from the cube and GNSS points. Finally, the GNSS points which had the shortest distance from each pixel location were identified by representing the point pairs in a matrix form. It was done by calculating Euclidean distance from each pair of pixels and GNSS coordinates. The information was preserved in a text file as comma-separated value (CSV) format, which contained the unique ID of the closest GNSS point for every pixel in the HSI cube.
Solar-Sensor Angle Calculation Overview of solar zenith angle or viewing zenith angle (VZA, θ V ), solar zenith angle (SZA, θ S ), sensor or viewing azimuth angle (VAA, φ V ), and solar azimuth angle (SAA, φ S ) calculations in terms of cross-grid UAV with push-broom hyperspectral sensor are illustrated in Figure 2c .
Solar Zenith Angle (SZA): The solar zenith angle (SZA) is similar to the VZA, but instead of the sensor as the moving vector, the position of the sun becomes the point of interest. SZA is a function of the raster location coordinates (longitude and latitude) and time of the day. SZA (θ S ) can be calculated from Solar Elevation Angle (α S ) using Equation 3 . θ S = 90 o -α S ( The pixel coordinates were attached with corresponding sensor points and each sensor point included the time information in UTC format. Also, the coordinates were converted from UTM to a geographic coordinate system (World Geographic System 1984) , so the values were available as latitude and longitude. A python package called PVLIB (v0.9.0) was used to calculate δ, h and eventually θ S for all pixel coordinates.
Solar Azimuth Angle (SAA): Solar azimuth angle (SAA) is a function of time and coordinate for each pixel location and can be calculated using Equation 8 . φ S = cos -1 [ sin(δ) cos(φ)-cos(δ) sin(φ) cos(h) cos(α S ) ] (8) where φ, δ, h and α S are the latitude, solar declination angle, hour angle and solar elevation angle, respectively.
Viewing Zenith Angle (VZA): The viewing zenith angle (VZA) is the angle between the vector from sensor and raster point (VR ⃗⃗⃗⃗⃗⃗ ), and the surface normal (Z ⃗ ⃗ ) from the raster point (also known as zenith), which can be defined as θ V . The UAV was flown at a 50 m altitude for the whole mission. Therefore, a perpendicular vector from the sensor point to the XY surface can be drawn as VV ⃗⃗⃗⃗⃗⃗ , where VV ⃗⃗⃗⃗⃗⃗ is 50 m. The angle between VR ⃗⃗⃗⃗⃗⃗ and RV ⃗⃗⃗⃗⃗⃗ is known as Viewing Elevation Angle (α V ) and can be calculated using Equation 1 . α V = tan -1 VV ⃗⃗⃗⃗⃗ RV ⃗⃗⃗⃗⃗ = tan -1 50 √(x v -x r ) 2 +(y v -y r ) 2 (1) where the coordinates of R and V are (x r ,y r ) and (x v ,y v ), respectively, calculated in a Universal Transverse Mercator (UTM) projection system. Therefore, RV ⃗⃗⃗⃗⃗⃗ can be calculated as the Euclidean distance between R and V ́. If α V is known, then θ V can be calculated using Equation 2 . θ V = 90 o -α V (2) For every pixel coordinate, corresponding θ V values were calculated and converted to degrees.
Viewing Azimuth Angle (VAA): Azimuth angle can be calculated in the XY plane, where it is the clockwise angle between a point of interest and the true north (Y ⃗⃗ ). For calculating the sensor or viewing azimuth angle (VAA, φ V ), an arbitrary north vector for any pixel coordinate (x r ,y r ) was created by adding 100 m to y r (Figure 2c2 ). The VAA can be calculated using Equation 7 . φ V = cos -1 [ a ⃗ b ⃗ |a ⃗ ||b ⃗ | ] ( 7 ) where a is the true north vector and b ⃗ is the vector between the raster point and corresponding sensor point.
RESULTS AND DISCUSSIONS The SZA (θ S ), SAA (φ S ), VZA (θ V ), and VAA (φ V ) were calculated for all 13 HSI cubes, but we will discuss only the 1 st HSI cube (Figure 3 ). Additionally, the descriptive statistics of the angles are provided in Table 2 . Table 2 . Descriptive statistics of the angles for the 1 st HSI cube.
Solar Angles The SZA and SAA shows different angular pattern in the resulting rasters (Figure 3b and 3c ). The SZA started decreasing along with the flight direction, whereas the SAA started to increase along with the flight direction. The total duration for capturing this HSI cube was 42.32 seconds, which resulted in low standard deviation for the angular values of SZA and SAA (Table 2 ). The verification of our calculation of solar angles was done by calculating the solar position based on National Oceanic and Atmospheric Administration (NOAA) Solar Calculator (NOAA 2021), which is an online tool to calculate approximate solar position in terms of coordinates and local time. To verify our result with the NOAA Solar Calculator, 5 randomly selected points were selected, and corresponding solar angles were extracted. Table 3 shows the SZA and SAA calculated based on NOAA Solar Calculator and our method, and the absolute differences observed. Calculator and our method. Abs. Diff. indicates the absolute differences between two methods and T is the order of points mentioned in Figure 3 . The SZA and SAA values calculated by NOAA Solar Calculator and our method showed slight differences at the decimal level. Since we used highly accurate PVLIB Python library to calculate the solar angles, we could provide coordinates and time information up to any decimal level possible. For instance, the time information in our method had 6 decimal places for second values. On the other hand, the NOAA calculator could only take the second values as integer. Moreover, NOAA (2021) indicates that due to the variations in the atmospheric conditions and uncertainty in the algorithms, there could be slight differences in the solar position calculations. These could be attributed to the slight differences in the solar angle values. However, having precise coordinate and time information in the angle calculation is highly preferable for remote sensing applications, specifically in HSI-based processing.
Sensor Angles The pattern of VZA (Figure 3d ) can be explainable in terms of the flight path. Since the flight path runs through the middle of the cube, the VZA values are close to zero near the flight path and starts increasing at the edge of the image. Since this is zenith angle from the sensor, there should be higher angles at the edge rather than the middle. The VAA shows comparatively larger range of angular values (Figure 3e ). Since azimuth angle is calculated as the clockwise angle from the north vector of each raster pixel, the right side of the cube resulted with smaller angular values, whereas the left side comprised of higher values. However, the pattern in the VAA raster may seem binary, but the inset map on Figure 3e shows an enlarged portion of the right side. The inset map shows that there exists angular variation along the flight path and the variation can be seen perpendicular to the flight line. Therefore, the standard deviation is the highest for VAA with larger range of angular values (Table 2 ).
Limitations The major issue encountered in this study was the lack of camera calibration. We used the vendor provided software (Headwall Spectral View v3.1.4) to ortho-rectify the HSI cubes. However, when the cubes were plotted in a GIS environment, it was noted that the overlapping regions from two consecutive HSI cubes did not exactly match. Therefore, the HSI cubes were georeferenced with a Light Detection and Ranging (LiDAR)-derived RGB point cloud using 6 control points. The LiDAR mission was also flown on the same days the HSI missions were performed. The LiDAR UAV point cloud was corrected using a GNSS base station established during the data collection time and the vendor provided software named, Phoenix LiDARMill (v2.0, Phonix LiDAR Systems, Texas, United States). After correction, the position accuracy was around ±0.1 cm. When the HSI cubes were georeferenced with the RGB point cloud, corresponding cubes matched properly with each other. However, the problem can be solved by performing a camera calibration. Probably the internal operating parameters (IOPs), boresight angles or the lever-arm offsets were changed from the initially approximated values by the vendor. LaForest et al., (2019) performed similar camera calibration technique to perform time-delay adjustment to similar type of HSI UAV platform that had accurate GNSS/IMU information. The same methodology can be applied to our study to improve upon the ortho-rectification of the HSI cubes as well. Therefore, careful considerations should be made when working with push-broom sensors and calibration flights should be conducted using randomly placed ground control points (GCPs) on the ground.
CONCLUSIONS The study demonstrates a simple methodology for calculating solar-sensor zenith and azimuth angles for a push-broom hyperspectral sensor equipped in a UAV. The results show that the method can deliver all the angles in raster format, which can be very helpful to perform BRDF corrections or radiative transfer model-based applications in remote sensing of vegetation. If this work is needed to be reproduced for other study areas using similar sensor and platform, then it will be easy to do so by utilizing the automatic Python workflow developed from this work. In future, we will improve the camera calibration issue incurred in this study and apply these angle rasters to perform radiative transfer modeling-based applications for plant phenotyping.
ACKNOWLEDGEMENT This work has been supported in part by NSF/USDA (2020-67021-31530), NASA (80NSSC20M0100), USGS AmericaView Grant (G18AP00077) and NSF Plant Genome Research Program (1733606). Figure 1 . 1 Figure 1. Study area and data collection instruments, (a) a RGB image of the maize field, (b) a close-up view of the field marked with a yellow box in (a), (c) the DJI M600 Pro UAV equipped with sensors, (d) a close up view of the sensor package which includes a Headwall Nano-Hyperspec VNIR camera, A FLIR Vue Pro thermal camera and APX-15 GNSS/IMU unit all attached in a DJI Gimbal, (e) the location of the study area, and (f) the cross-grid flight pattern created in UGCS flight planning software (v4.0.187) which was used for data collection.
Figure 2 . 2 Figure 2. Overview of methods for the extraction of solar-sensor zenith and azimuth angles, i.e., (a) hyperspectral cube processing involves converting digital number (DN) to radiance, reflectance, and ortho-rectified images, (b) locating corresponding sensor location for each pixel in a hyperspectral cube, and (c) calculating three solar-sensor zenith and azimuth angles.
3) sin α S = sin φ sin δ + cos φ cos δ cos h (4)where, φ is the latitude of the location, δ is the solar declination angle and h is hour angle. Declination angle (δ) is the angle between the line joining the centers of the Sun and the Earth and its projection on the equatorial plane. The value of δ can range from -23.44 to 23.44 and calculated using Equation5, where d is the number of days since the beginning of the year. Hour angle (h) is the position of the sun relative to solar noon and can be calculated using Equation6, where LST is the local solar time. Solar hour angle is 0 at solar noon and it increases by 15 after each hour.
Figure 3 . 3 Figure 3. Resulting angles for the 1 st HSI cube, where (a) RGB true color composite, (b) solar zenith angle (θ S ), (c) solar azimuth angle (φ S ), (d) viewing zenith angle (θ V ), and (e) viewing azimuth angle (φ V ). The black dots (in b and c) are test points that were verified with alternative source. The inset map in the VAA (e) shows a small zoomed-up portion, which indicates the changing angular pattern in VAA.
Table 1 . 1 Sensor specifications Sensor Specifications Headwall Wavelength (nm) 400 -1000 Nano Spatial bands 640 Hyperspec Spectral bands 269 VNIR Field of View () 50.684 Focal length (mm) 12 Dimension (mm) 76(L)×76(W)×119(H) Weight (g) 680 APX-15 Channels 336 GNSS/IMU Dimension (mm) 67(L)×60(W)×15(H) Weight (g) 60
Table 3 . 3 Comparison of solar angles between NOAA Solar
This contribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper.https://doi.org/10.5194/isprs-annals-V-3-2022-131-2022 | Author(s) 2022. CC BY 4.0 License.
This contribution has been The double-blind peer-review was conducted on the basis of the full paper.https://doi.org/10.5194/isprs-annals-V-3-2022-131-2022 | Author(s) 2022. CC BY 4.0 License.
|
10.1056/nejm188512241132607
|
public-domain
| null | null |
openalex
|
later a cyclitis developed which soon subsided. In December, Sn. 1 was read, though there were opacities in the vitreous. During the next six months Miss X.. was kept indoors by sciatica. She read and sewed at will, and later was able to exercise in her garden. September 14th. Feeling very well, she went out to drive at 11 a.m., (eleven months after the extraction.) The day was bright, and she said she looked pretty steadily at the various objects that came into view and with absorbing interest and enjoyment till her return at 1 p.m. On leaving her carriage everything around her suddenly took on a dark-pink color, and this redenvironment continued after she had entered her house, it persisted after the lamps had been lighted though of a different shade and this distressing red vision re- mained unaltered for six days. Although living but a few blocks away, she delayed consulting me till the 21st, (feeling doubtful, very likely, if she should get any relief?), when the light was less annoying, though all objects still seemed pink. The eye was not inflamed and the sight was better than in December. At the end of another week the erythropsia had entirely disappeared, having faded grad- ually away. As the patient has again been confined to her house, it is uncertain whether her trouble will return when she is again subjected to the full light of day. SOME FACTS BEARING ON THE \l=AE\TIOLOGY OF CARCINOMATOUS DISEASES. BY G. A. WHEELER, M.D., CASTINE, MAINE. The predisposing causes of cancer are stated by nearly all authors who have written about this disease, as unknown. It is usually admitted, however, that all causes which tend to lower the vitality of the system are among the number, and by many there is thought to be a kinship between tuberculous and carcinoma- tous affections. As phthisis undoubtedly bears a cer- tain relation to the humidity or dryness of the soil, the query is forced upon us whether carcinoma may not also have a similar relation either to the humidity or to the composition of the soil. I am led to these reflections by a consideration of the cases of malignant disease which were presumably cancerous, though not actually demonstrated to be such by the microscope, which have occurred within the last eighteen years, in the small village of Castine, Maine, and in a particular part of the town. In fact, nearly all on one particular street, or quite near to it. The few other cases mentioned, having at some previous time lived on or near this street. The soil of this portion of the village is superficially a sandy loam, beneath which is a layer of coarse gravel and still deeper a blue clay. All the drinking water upon the street is extremely hard but no analy- sis of it has ever been made. The street commences near the .summit of a hill about two hundred feet above the level of the ocean and slopes down to the sea. The drainage is excellent. In the accompanying plan of the village the houses where the cases about to be mentioned occurred, are numbered to correspond with those cases. 1. N. G., male, age about fifty. Lived in the house for many years. No hereditary predisposition known. Occupation, a ship carpenter. Autopsy showed seirrlius of the cardiac orifice of the stomach. Death from starvation. 2. J. N., male, age about fifty. Occupation, ship carpenter. No hereditary predisposition known. Had a tumor of the thigh for which he was admitted to the Massachusetts General Hospital, but for which noth- ing was done there. He afterwards went to the Bos- ton Homoeopathic Hospital, where he was operated upon, but the tumor returned, and he subsequently died, and the death was reported as from cancer. Had lived in this house for years. 3. Wife of the above. Age about forty-five. She was attended by the writer a few weeks before her death, which was due to malignant disease of the womb. 4. C. G, male. Age about fifty. Merchant. No hereditary tendency. Had occupied the house for years. He was operated upon unsuccessfully by the late Dr. Greene, of Portland, for scirrhus of the rectum. 5. S. H., female, single. Age sixty-nine. No he- reditary tendency. Had occupied the house for years. Had a scirrhus of the breast removed at the Maine General Hospital. Died from constitutional infection. Had also a large abdominal tumor. 6. J. II. N., male. Carpenter. Age about sixty. Had lived, in the house from childhood. His mother is said to have had a "sore" cancer of the face. Autopsy showed scirrhus of the pylorus. 7. B. K., female, widow. Age about fifty-five. Had lived in this house and in town only two years. No hereditary tendency known. Died from carcinoma uteri. Is supposed to have had the disease when she came to town, but this is a matter of doubt. Her sister died in the same house two years later of phthisis. 8. B., female, married. Age about sixty-live. No hereditary tendency. Had an abdominal tumor which from its hard, knobby feel I diagnosticated as scirrhus. Had marked cachexia when she died. No autopsy. Had lived in this house for years. 9. D. L., female, married. Age about sixty-five. No hereditary tendency known. Had resided in this house for years. Diagnosis, carcinoma uteri. Was under the charge of Dr. Stevens, now deceased. 10. M. G, female, widow. Age about eighty. No hereditary tendency. Had carcinoma mamma'. Had resided in this house for years. 11. F. G, female, married. Age forty-four. Cauli- flower excrescence of cervix uteri. Removed by Or. F. F. Sauger and myself. Disease extended and fin- ally destroyed recto-vaginal septum. No hereditary tendency. Had lived in this house twenty years. 12. G. T., male. Age fifty-five. Trader. Died from a malignant disease affecting the stomach, and probably involving other organs. No autopsy. No hereditary tendency, Had occupied this house for ten or twelve years but had lived in the one opposite 4 and 5, for a still longer period. 13. S. D., female, married. Age about forty-five. No hereditary tendency known. Died from what was diagnosticated by her attending physician as carcinoma uteri. She had lived in this house for several years. but had also lived many years in the one marked 5. Had a child die of phthisis. il. L. W., female, widow. Age thirty-seven. Mother died of heart disease, but hada suspicious tu- mor of the abdomen. This patient was operated upon by Dr. E. F. Sänger and myself for cauliflower excres- cence of cervix uteri. Is still living, but is likely, to succumb to the disease eventually. This patient has lived in this house for many years, but formerly lived two houses from the one marked G. According to common report there have been one or two other cases of cancer in this same locality, but I have been unable to verify the fact. I hayre one pa- tient on hand, however, who resides just above 4, who has had a polypus uteri removed, and who now has a large uterine tumor of some kind, the nature of which is still in doubt, but which I am fearful may prove to be carcinomatous. The surprising thing about these cases is that they should have been confined to such a limited territory. After a residence of fifteen years in this town I have not known and am unable to learn of any other cases of presumed carcinoma occurring in any other portion of this village, and but few cases have occurred in my practice elsewhere. If there be not some local cause for these cases it is remarkable that they should have occurred where they did. I am aware that so limited a number of cases can have no great weight towards establishing a local cause, but it seems to me they should have some, and they are given in the hopes of inducing other physicians to pay some attention to the localities in which their cases occur. Report s of Societies. Dr. Abbott, in opening the debate, said that some years since, he had a case of hydatid mole similar to that reported by Dr. Dunn. When he reached the woman, he found that the mass had come away entire, and consisted of a great number of cysts, varying from the size of a pea to a plum, and in quantity, he should judge, more than a pint. There was no trace of a foetus, and no haemorrhage followed the delivery. The mass was passed, he believed, early in pregnancy, and there were no bad symptoms before or after the delivery. This yvas the only case of uterine hydatids he had ever seen. Dr. Doe remarked that he had a case similar to that mentioned by Dr. Abbott. A large mass of cysts, suf- ficient to fill a six-quart pail, came away at about the seventh month. No foetus was discovered. De. A. E. McDonald mentioned the following case : Mrs. IL, aged twenty-five years, multípara ; family history^good ; no syphilis. She came under my care about ten years ago, and yvas treated with sat- isfactory results for uterine displacement and chronic en- do-mitritis. About a year afterward, she again consulted me, stating that she had not menstruated for a period of four months previously, but was then having slight sanguineous discharge, with occasional pain in the region of the uterus. Her abdomen appeared unusually large for that of a woman in the fourth month of ges- tation, but I thought she might have made a mistake as to the date of her conception. In view of her con- dition, I did not deem a critical examination advisable. Restin the recumbent position was advised, with medi- cine to relieve pain. I was summoned on the following day, and found her having quite strong expulsive pains at short intervals. My attention was called to the chamber-vessel which she had used a short time before my arrival, and 1 found in it about a gill of saccules attached to each other, and which had come away during one of her expulsive efforts while on the vessel. These saccules were about the size of small grapes, and semi-transparent, corresponding somewhat to Gooch's simile of " White currants floating in red cur- rant juice." My finger, on introducing it into the vagina, came in contact with a large, soft mass, which, to the feel, resembled a recent blood-clot, excepting that it was more spongy. I succeeded in removing the mass in its entirety, and found it to be a true mole of the vesicular or hydatidiform variety. I discovered no trace of a foetus in the mass, that having disappeared in the earlier stages of gestation. The whole quantity which came away would more than lili a pint measure. There was -no haemorrhage to contend with in the case, and the patient made a speedy and perfect recovery. Since then, she has given birth to two healthy children who are now living, aged respectively five and three The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at NYU WASHINGTON SQUARE CAMPUS on June 24, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright 2010 Massachusetts Medical Society.
SUFFOLK DISTRICT MEDICAL SOCIETY. SECTION OF OBSTETRICS AND GYNAECOLOGY. ROBERT B. DIXON, M.D., SECRETARY. November 18, 1885, Dr. James R. Ciiadwick in the Chair. Dr. W. A. Dünn read a paper entitled IIYDATIDIFORM MOLE OF THE UTERUS.1
. W. Driver remarked that during a large 1 See page 612. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at NYU WASHINGTON SQUARE CAMPUS on June 24, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright 2010 Massachusetts Medical Society.
|
10.1371/journal.pone.0230645
|
cc-by
| null | null |
openalex
|
Houttuynia cordata Thunb. has been used as a traditional medicine to treat a variety of ailments in Asian countries such as China, Japan, South Korea, and Thailand. In Thailand, H. cordata fermentation products (HCFPs) are commercially produced and popularly consumed throughout the country without experimental validation. Anti-inflammatory activity of H. cordata fresh leaves or aerial parts has previously been reported, however, the antiinflammatory activity of the commercially available HCFPs produced by the industrialized process has not yet been investigated. The aim of this study was to evaluate in vitro and in vivo anti-inflammatory potential of the selected industrialized HCFP. LPS-induced RAW264.7 and carrageenan-induced paw edema models were used to evaluate the antiinflammatory activity of HCFP. The phenolic acid components of HCFP aqueous and methanolic extracts were investigated using HPLC analysis. In RAW264.7 cells, the HCFP aqueous and methanolic extracts reduced NO production and suppressed LPS-stimulated expression of PGE 2 , iNOS, IL-1β, TNF-α and IL-6 levels in a concentration-dependent manner, however, less effect on COX-2 level was observed. In Wistar rats, 3.08 and 6.16 mL/kg HCFP reduced paw edema after 2 h carrageenan stimulation, suggesting the second phase anti-edematous effect similar to diclofenac (150 mg/kg). Whereas, 6.16 mL/kg HCFP also reduced paw edema after 1 h carrageenan stimulation, suggesting the first phase antiedematous effect. Quantitative HPLC revealed the active phenolic compounds including syringic, vanillic, p-hydroxybenzoic and ferulic acids, which possess anti-inflammatory activity. Our results demonstrated for the first time the anti-inflammatory activity of the industrialized HCFP both in vitro and in vivo, thus validating its promising anti-inflammation potential.Introduction Inflammation is a host response against infection, foreign stimulant and tissue injury. Although inflammation is a process of the immune response in our body, it can damage the body when it is out of control. While acute inflammation is a normal part of the defense response, chronic inflammation is a complex process stimulated by activating inflammation or immune cells. During the inflammatory process, many types of cells are activated, and these cells secrete various pro-inflammatory mediators, including cytokines (IL-1β, TNF-α, IL-6), nitric oxide (NO) and prostaglandin E2 (PGE 2 ) . Overproduction of inflammatory mediators leads to chronic inflammation, which can cause many diseases such as rheumatoid arthritis, cancer and allergies . During the inflammatory response, immune cells are also activated by adhesion molecules-activated signals to increase the migration capacity to inflamed tissue and finally to form heterotypic cell clustering between the immune cells, endothelial cells, and inflamed cells. Indeed, various inflammatory stimuli such as LPS and proinflammatory cytokines activate immune cells to up-regulate such inflammatory states [2, 3] . Hence, these cells are useful targets for developing new anti-inflammatory drugs and exploring the molecular anti-inflammatory mechanisms of a potential drug. Many drugs have been developed to treat inflammation and nociceptive symptoms however, undesired adverse effects of these clinical anti-inflammatory drugs have consistently evidenced . Non-steroidal anti-inflammatory drugs (NSAIDs) are normally used for the treatment of pain and inflammatory conditions, however, many NSAIDs are associated with undesired side effects including congestive heart failure, bleeding of the gastrointestinal tract and chronic kidney disease . Therefore, the search for alternative substitutes such as plantderived anti-inflammatory agents with the ease of availability and fewer side effects is urgently required to develop safe drugs for clinical use. Houttuynia cordata Thunb. is a perennial herbaceous plant mostly distributed in East Asia, and generally grown for local vegetable consumption in the North and Northeast of Thailand. H. cordata has been used as a medicinal plant possessing many biological properties including antioxidant, anticancer, and anti-inflammatory activities . As a traditional medicine in China, H. cordata has been used to treat ulcers, sores, heatstroke, diarrhea, and dysentery . In Korea, it has been used for the treatment of pneumonia, bronchitis, dysentery, dropsy, uteritis, eczema, herpes simplex, chronic sinusitis and nasal polyps . In Thailand, it has been used as an immunostimulant herb and anticancer agent . Nowadays, H. cordata is considered for a high-value industrial crop in Thailand and it has been fermented with probiotic bacteria to yield a H. cordata fermentation product (HCFP) commercially available. The microbial fermented herbal plant is a promising alternative source for many flavonoid molecules including anthocyanins, flavones and flavanones . Probiotics are microorganism exerting health-promoting functions in humans and animals , improving the nutraceutical value of the herbal plant products by breaking down undesirable phytochemicals, and producing certain desirable compounds . The fermentation process has increased the flavonoid content of H. cordata fermentation products conferring excellent anti-inflammatory effects in LPS-stimulated cells . Accordingly, many HCFPs have been commercially distributed and popularly consumed throughout Thailand. Previous studies reported that the industrial process caused a reduction in phenolic content of natural products [14, 15] , which may affect their biological properties. Anti-inflammatory activity of H. cordata fresh leaves or aerial parts has previously been studied [16, 17, 18] , however, the anti-inflammatory activity of HCFPs produced by industrialized process has not yet been investigated. Therefore, we aimed at investigating the anti-inflammatory activity of the industrial HCFP in LPS-stimulated RAW264.7 cells as well as its phenolic acid content to provide information for the general public or consumers. Here, we demonstrated the phenolic acid profiles and antiinflammatory activities of aqueous and methanolic extracts of the industrial HCFP Dokudami manifested by inhibiting the production of NO, PGE 2 and inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Furthermore, the anti-inflammatory activity of this industrialized product was also confirmed using the rat paw assay.
Materials and methods
Materials The dietary supplement H. cordata fermentation product (HCFP), Dokudami, was obtained from the Prolac (Thailand) Co., Ltd., Lamphun, Thailand. The information on plant ingredient and serving suggestion of the HCFP were obtained from the label on its container. The major ingredients of this HCFP are composed of 99.3% (w/w) aerial parts of H. cordata and 0.7% (w/w) sugar cane powder. Serving suggestion is as follows: 5-15 ml twice a day in the morning before bedtime and before meal. H. cordata was cultivated by the Prolac (Thailand) Co., Ltd. in an organic farm in Chai Badan district, Lopburi province, Thailand. The fermentation product Lot no. 14/5/2015 was used throughout the study. RAW264.7 cells were obtained from Dr. Pramote Mahakunakorn, Faculty of Pharmaceutical Science, Khon Kaen University, Thailand. Male Wistar rats (250-300 g) were obtained from registered animal breeders, Nomura Siam International Co., Ltd., Bangkok, Thailand. LPS (E. coli 0111: B4) and diclofenac sodium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Griess reagent for nitrite determination was purchased from Molecular Probes (Invitrogen, USA). All antibodies used in this study were purchased from Cell Signaling (USA). PGE 2 EIA was purchased from ANOVA (Taiwan). The ELISA kits for measuring cytokines (IL-1β, TNF-α, IL-6) were purchased from BioLegend (California). RPMI 1640 medium, fetal bovine serum (FBS), trypsin-EDTA and penicillin/streptomycin were obtained from Gibco/Invitrogen Crop. (Grand Island, NY, USA).
Cell culture and animals RAW264.7 macrophage cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and incubated at 37 ̊C in an atmosphere containing 5% CO 2 . Wistar rats were recovered from transportation for 1 week before the study. The rats were maintained at Northeast Laboratory Center, Khon Kaen University, Thailand. Details of animal welfare are as follows: Shelter: case size 37.5 x 48 x 18.5 cm (wide x length x high), with sterilized-wood shavings for bedding, Food: sterilized commercial food, Water: reverse osmosis (OR) with choline 3-4 ppm, Environment enrichment: social housing, free excess food, and water, Environment: temperature: 23±2 ̊C, humidity: 30-60% RH, dark: light cycle: 12:12 h, illumination: 350-400 Lux, ventilation: 10-15 ACH, noise: no exceed 85 Decibels. The experimental procedure was approved by the Institutional Animal Care and Use Committee of Khon Kaen University, based on the Ethic of Animal Experimentation of National Research Council of Thailand. The approval number was IACUC-KKU-101/60.
Preparation of the lyophilized powder of HCFP aqueous extract To obtain polar phytochemical compounds, 50 mL of HCFP was centrifuged at 2,815 x g for 15 min and the supernatant (aqueous fraction) was filtered through Whatman grade No. 4 filter paper. The filtrate containing water-soluble constituents was lyophilized to obtain a lyophilized powder (aqueous extract). The extraction yield was 13.80 ± 0.57 mg/mL. The HCFP lyophilized powder was re-dissolved in double distilled water to obtain desired concentrations.
Preparation of HCFP methanolic (phenolic-rich) extract To obtain both polar and nonpolar phenolic compounds in the free forms, 140 mL of methanol was added to 60 mL of HCFP and then the mixture was stirred for 2 h at room temperature. The filtrate was evaporated to 60 mL by rotary evaporator, then added with 60 mL of 2 M NaOH and stirred continuously for 12 h at room temperature. The mixture was centrifuged at 1,700 x g for 20 min and then filtered through Whatman grade No. 4 filter paper. The supernatant was repeatedly extracted three times with 80 mL of diethyl ether and the aqueous phase was collected and the diethyl ether phase was discarded. The aqueous phase was adjusted to pH 1.5 by 10 M HCl and filtered through Whatman grade No. 4 filter paper. The filtrate was extracted further with 80 mL of diethyl ether for three times, in which the portion of diethyl ether was collected. Sodium sulphate (Na 2 SO 4 ) anhydrous was used to dehydrate the diethyl ether phase, which was then filtered through the filter paper. A rotary evaporator was used to evaporate the filtrate to 5 mL, which was then finally evaporated to dryness under a gentle stream of nitrogen gas. The extraction yield was 5.36 ± 0.96 mg/mL.
Cell viability assay The viability of RAW264.7 cells was determined colorimetrically using 3-(4,5-dimethylthiazolyl)-2-2,5-diphenyltetrazolium bromide (MTT) reagent (Invitrogen, USA). The cells at a density of 8 x 10 3 cells/well were seeded in 96 well plates. After 24 h, various concentrations of HCFP aqueous (5-1,500 μg/mL) and methanolic (4-18 μg/mL) extracts were added to the cells and incubated for 24 h. The MTT solution was added to each well and incubated for 2 h at 37 ̊C. After removing the solution, each well was added with DMSO to dissolve the formazan dye. The absorbance of formazan was measured using the microplate reader (Bio-Rad, USA) at 550 nm and 655 nm as a reference wavelength for subtraction of optical density caused by cell debris.
Nitrite determination The nitrite concentration in the culture medium of treated and untreated RAW264.7 cells was measured as an indicator of NO production according to Griess reaction . Briefly, the cells (1x10 5 cells/well) were seeded into 24-well plates for 24 h, and then pre-treated cells with various concentrations of HCFP aqueous (25-750 μg/mL) and methanolic (4-12 μg/mL) extracts for 2 h. After 2 h incubation, the cells were incubated with LPS (1 μg/mL) for 24 h. The treatment with diclofenac (DCF; 25 μg/mL) was used as a positive control. The cultured medium was then collected and mixed with an equal volume (1:1) of Griess reagent (Invitrogen, USA). After 10 min incubation at room temperature, the absorbance at 550 nm was measured using a microplate reader.
Prostaglandin E2 (PGE 2 ) determination The PGE 2 metabolite is measured by using an enzyme immunoassay (EIA) kit (Abnova, Taiwan) based on the conversion of all major PGE 2 metabolite into a single stable derivative. The cells (1 x 10 5 cells/well) were seeded into 24-well plates and cultured for 24 h. The cells were pre-treated with various concentrations of HCFP aqueous (25-750 μg/mL) and methanolic (4-12 μg/mL) extracts for 2 h and thereafter incubated with LPS (1 μg/mL) for 24 h. The treatment with diclofenac (DCF; 25 μg/mL) was used as a positive control. Subsequently, PGE 2 concentration in a culture medium was determined with PGE 2 EIA kit according to the manufacturer's instructions.
Western blot analysis RAW264.7 cells (1 x 10 6 cells) were seeded into a 5.5-cm culture dish and cultured for 24 h. Cells were pre-treated with various concentrations of HCFP aqueous (25-750 μg/mL) and methanolic (4-12 μg/mL) extracts for 2 h and then incubated with LPS (1 μg/mL) for 24 h. The treatment with diclofenac (DCF; 25 μg/mL) was used as a positive control. The treated cells were harvested and lysed with lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 1 h on ice. The protein concentration was determined by Bradford protein assay (Bio-Rad, USA). Equal amounts of protein (30 μg) were loaded and separated on 12% SDS-polyacrylamide gel and afterward the proteins were transferred to the PVDF membrane. The membrane was blocked with a blocking solution, 5% skim milk in phosphate-buffered saline containing Tween-20 (PBST), for 1 h at room temperature, and then incubated with monoclonal anti-iNOS, anti-COX-2, anti-β-Actin (1:1000 dilutions, Cell signaling, Germany) for overnight at 4 ̊C. The blots were washed twice with PBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilutions, Cell signaling, Germany) for 2 h at room temperature. Blots were washed again twice with PBST and PBS, respectively. The protein bands were visualized using ECL detection reagent (GE healthcare, UK).
RNA isolation and RT-PCR analysis Cells (1 x 10 6 cells) were seeded into a 5.5-cm culture dish and cultured for 24 h. Cells were pre-treated with various concentrations of HCFP aqueous (25-750 μg/mL) and methanolic (4-12 μg/mL) extracts for 2 h and then incubated with LPS (1 μg/mL) for 6 h. Total RNA from treated cells was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol and the RNA was kept at -70 ̊C until used. Total RNA (1 μg) was used for reverse transcription reaction using M-MuLV reverse transcriptase (NEB, UK), 0.5 μM specific reverse primer, deoxyribonucleotide triphosphate (dNTP, 0.2 mM) and 1 U RNase inhibitor. The reaction was incubated at 42 ̊C for 1 h and the M-MuLV reverse transcriptase was then inactivated by heating at 65 ̊C for 20 min. The PCR reactions were carried out in a total volume of 25 μl containing 2.5 U of Taq DNA polymerases, 0.2 mM dNTP, 1X reaction buffer, and 0.5 μM of forward and reverse primers as listed in S1 Table . After initial denaturation for 30 sec at 95 ̊C, the amplification by 30 cycles of 94 ̊C for 45 sec (denaturing), 50-55 ̊C for 45 sec (annealing), 72 ̊C for 45 sec (extension), was carried out. The PCR products were analyzed by 1.5% agarose gel electrophoresis. The level of mRNA expression was quantitated by Quantity One software 4.4.1 (Bio-Rad) using β-actin band intensity as the internal control.
Determination of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) Cells (1 x 10 5 cells/well) were seeded into 24 well plates and cultured for 24 h, and then pretreated with various concentrations of HCFP aqueous (25-750 μg/ml) and methanolic (4-12 μg/mL) extracts for 2 h. Thereafter, the cells were incubated with LPS (1 μg/mL) for 24 h. The levels of these cytokines in the cultured medium of treated RAW264.7 cells were quantified using ELISA kits according to the manufacturer's instructions. The absorbance at 450 nm was measured using the fluorescence microplate reader (SpectraMax M5, Molecular Devices, USA), and 570 nm was used as a reference wavelength.
In vivo experiment Animal studies were performed in obligation with the Institutional Animal Care and Use Committee at Khon Kaen University, Khon Kaen, Thailand (Approval ID: AE101/60) and were performed according to guidelines established by the Ethical Principles and Guidelines for the Use of Animals for scientific purposes, National Research Council of Thailand. All experiments were carried out with six animals in each group. In this study, carrageenan-induced inflammation in the rat paw was used as a model system for in vivo anti-inflammatory study. Male Wistar rats (250-300 g) were divided into four different groups, (1) negative control (carrageenan-treated), (2) HCFP 1 (concentration 1-treated), (3) HCFP 2 (concentration 2-treated), and (4) positive control (Diclofenac-treated). HCFP (3.08 and 6.16 mL/kg) and Diclofenac (150 mg/kg) were administered by orally 1 h before carrageenan induction. The rats received a sub-plantar injection of 100 μL of 1% (w/v) suspension of carrageenan lambda in the right hind paw. The volume of rat paw in all animals was measured at 1, 2 and 3 h after carrageenan injection by using Plethysmometer (Ugo Basile Model 7140, Italy). After λ-carrageenan injection and measurement the volume of rat paw at three hours, the rats were sacrificed by injecting pentobarbital sodium anesthetic. The results were expressed as the changes in paw volume from the baseline value. The percentage of paw edema was calculated using the following equation: %Paw edema 1⁄4 ðV À ViÞ 100=Vi Where V = Paw thickness after carrageenan injection and Vi = Paw thickness at 0 time.
HPLC analysis Phenolic acid compositions in HCFP aqueous and methanolic extracts were analyzed by using reverse-phase HPLC as previously described , with some modifications. The columns used to identify phenolic acids in HCFP aqueous and phenolic extracts were Inertsil 1 -ODS-4 C18 column (4.6 mm i.d. x 250 mm, 5 μm particle size) and Waters system C18 column (3.9 mm i. d. x 150 mm, 5 μm particle size), respectively, due to availability of the columns at Facilities Service Center, Faculty of Science, Khon Kaen University, Thailand. The linear gradient of solvents A (100% acetonitrile) and B (1% acetic acid in deionized water) for Inertsil 1 -ODS-4 C18 column was as follows: 0 min, 3% A: 97% B; 5 min, 8% A: 92% B; 15 min, 8% A: 92% B; 25 min, 10% A: 90% B; 55 min, 10% A: 90% B. The linear gradient of solvents for Waters system C18 column was as previously described . The internal standard (m-hydroxybenzaldehyde; 1 μg) was used to ensure the accuracy of phenolic acid identification.
Statistical analysis Data are expressed as mean ± S.D. form two or three independent experiments. The data analysis was performed by one-way ANOVA with Duncan's post hoc test. Differences were considered to be significant at p < 0.05.
Results
Effect of HCFP aqueous and methanolic extracts on cell viability in RAW264.7 cells To study the effect of HCFP aqueous and methanolic extracts on inflammatory responses in vitro, RAW264.7 macrophage cells, which play an important role in the maintenance of tissue homeostasis, were used as a model system. The concentrations of both extracts that had no adverse effects on the growth of RAW264.7 cells were determined using MTT assay. Both HCFP aqueous (Fig 1A ) and methanolic (Fig 1B ) extracts showed no toxicity against RAW264.7 cells at the concentration ranges of 5-750 and 4-12 μg/mL, respectively. The cell viability of more than 90% as compared with a control group was considered non-toxic. Thus, these concentration ranges of HCFP aqueous and methanolic extracts were selected for further study on the anti-inflammatory effect.
Effect of HCFP aqueous and methanolic extracts on nitric oxide (NO) production of LPS-stimulated RAW264.7 cells NO is a pro-inflammatory mediator produced by activated macrophages that induce inflammation under pathological conditions . To investigate the effect of HCFP aqueous and C, D ) and PGE 2 levels (E, F) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were incubated with aqueous (5-1,500 μg/mL) and methanolic (4-18 μg/mL) extracts for 24 h. Cell viability was assessed by MTT assay. The results were reported as a percentage of cell viability compared with untreated controls and expressed as mean ± S.D. of three independent experiments. For determinations of NO production and PGE 2 levels in LPS-stimulated RAW264.7 cells, the cells were pre-treated with indicated concentrations of aqueous (C, E) and methanolic (D, F) extracts for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. The nitrite production and PGE 2 levels in cultured medium were determined by using Griess reagent and PGE 2 EIA kit, respectively. Statistically significant inhibition of NO production and reduction of PGE 2 levels ( p < 0.05) were found as compared with the LPS group. Data were obtained from three and two independent experiments, respectively. https://doi.org/10.1371/journal.pone.0230645.g001 methanolic extracts on NO production, RAW264.7 cells were pre-treated with aqueous (25-750 μg/mL) and methanolic (4-12 μg/mL) extracts and thereafter stimulated with LPS (1 μg/ mL). NO production was determined by the measurement of nitrite released into the cultured medium using the Griess reagent. The NSAID drug diclofenac (DCF; 25 μg/mL), a positive control for comparing the activity of HCFP extracts, inhibited NO release by 75.40% in LPSstimulated RAW264.7 macrophages (Fig 1C and 1D ). The maximum (750 μg/mL) and minimum (25 μg/mL) concentrations of the HCFP aqueous extract reduced NO production by 74.40% and 35.22%, respectively (Fig 1C ). Notably, the HCFP methanolic extract at maximum (12 μg/mL) and minimum (4 μg/mL) concentrations reduced NO production by 62.26% and 12.66%, respectively (Fig 1D ). Accordingly, our results showed that both HCFP aqueous and methanolic extracts inhibited NO production in a concentration-dependent manner in LPSstimulated RAW264.7 cells.
Effect of HCFP aqueous and methanolic extracts on PGE 2 production in LPS-stimulated RAW264.7 cells PGE 2 produced from arachidonic acid through the function of cyclooxygenase (COX) enzymes during inflammatory responses exacerbates the inflammatory process through several signaling modules . We sought to investigate the inhibitory effect of HCFP extracts on PGE 2 levels in LPS-stimulated macrophages, which may be an effective strategy for treating inflammatory disorders. Similar to the effect on NO production, both HCFP aqueous and methanolic extracts caused a dose-dependent inhibition of PGE 2 production in LPS-stimulated RAW264.7 cells (Fig 1E and 1F ). PGE 2 level was increased to 3,503.11 pg/mL in LPS treatment, whereas in the absence of LPS, PGE 2 level was reduced to 179.42 pg/mL. PGE 2 levels were significantly reduced in the cells treated with aqueous (25-750 μg/mL) (Fig 1E ) and methanolic (4-12 μg/mL) (Fig 1F ) extracts, especially at the highest concentrations tested (58.59% and 51.00% reduction by aqueous (750 μg/mL) and methanolic (12 μg/mL) extracts, respectively). However, diclofenac (25 μg/mL) inhibited PGE 2 production by 93.04% in LPSstimulated RAW264.7 macrophages (Fig 1E and 1F ).
Effect of HCFP aqueous and methanolic extracts on expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both mRNA and protein expression levels in LPS-stimulated RAW264.7 cells NO is produced from the conversion of L-arginine to L-citrulline by iNOS , whereas PGE 2 production is mediated by COX-2 . We sought to investigate whether the observed inhibition of HCFP aqueous and methanolic extracts on LPS-induced NO and PGE 2 production (Fig 1C -1F ) was related to the modulation of iNOS and COX-2 using RT-PCR and Western blot analysis. The mRNA and protein expression levels of iNOS and COX-2 were minimally detected and undetectable, respectively, whereas their levels were significantly increased by LPS treatments (Figs 2 and 3 , respectively). iNOS mRNA induction was significantly suppressed by pre-incubation with the NSAID drug diclofenac (25 μg/mL), however, the greater suppression was observed for pre-incubation with both HCFP aqueous (25-750 μg/mL) (Fig 2A and 2B ) and methanolic (6-12 μg/mL) extracts (Fig 2C and 2D ). iNOS protein level was significantly decreased for pre-incubation with both HCFP aqueous (25-750 μg/mL) (Fig 3A and 3C ) and methanolic (4-12 μg/mL) (Fig 3B and 3D ) extracts. Pre-incubation with diclofenac did not cause a significant decrease in either mRNA (
Effect of HCFP aqueous and methanolic extracts on expressions of IL-1β, TNF-α and IL-6 at both mRNA and protein expression levels in LPSstimulated RAW264.7 cells Interaction between LPS and the membrane receptor CD14 of macrophages caused the induction of pro-inflammatory cytokines including IL-1β, TNF-α and IL-6 . These pro-inflammatory cytokines have been considered as targets for anti-inflammatory therapies . To investigate the anti-inflammatory action of aqueous and methanolic extracts of H. cordata fermentation product, the production of the pro-inflammatory cytokines was evaluated by both RT-PCR and ELISA. The mRNA levels of IL-1β, TNF-α, and IL-6 were up-regulated in LPS treated cells compared with untreated controls (Fig 2A -2D ). Diclofenac treatment caused a significant decrease in mRNA levels of IL-1β and TNF-α but not IL-6 (Fig 2A -2D ), whereas treatments with both HCFP aqueous (25-750 μg/mL) (Fig 2A and 2B ) and methanolic (4-12 μg/mL) (Fig 2C and 2D ) extracts caused a significant decrease in IL-1β, TNF-α, and IL-6 mRNA levels. Based on ELISA results, induction of IL-1β, TNF-α, and IL-6 protein levels was dose-dependently suppressed by pre-incubation with both HCFP aqueous (25-750 μg/mL) (Fig 4A , 4C and 4E ) and methanolic (4-12 μg/mL) (Fig 4B , 4D and 4F ) extracts. LPSinduced IL-1β production was inhibited by 68.78 or 62.09% when treated with the highest
Effect of HCFP on carrageenan-induced paw edema in Wistar rats To evaluate the in vivo anti-inflammatory activity of the HCFP Dokudami, the carrageenaninduced paw edema model was chosen as it is sensitive and reproducible in vivo test for NSAID drugs and has long been established as a valid model for studying new anti-inflammatory drugs . The formation of paw edema was gradually increased within the first hour after carrageenan injection (Fig 5 ). A common clinical NSAID drug diclofenac (DCF) was used as a positive control pre-treated at 150 mg/kg. DCF significantly (p < 0.05) reduced paw edema after 2 h carrageenan stimulation. Similarly, the HCFP (3.08 and 6.16 mL/kg) also significantly (p < 0.05) reduced paw edema after 2 h carrageenan stimulation. However, pretreatment of HCFP at a concentration of 6.16 mL/kg caused a significant (p < 0.05) reduction of paw edema after 1 h carrageenan stimulation. 1 ). Whereas, seven phenolic acids were identified in methanolic extract of HCFP including protocatechuic, p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids (Fig 6D and Table 1 ). Among the identified phenolic acids of HCFP, the most abundant phenolic acid in both aqueous and methanolic extracts was syringic acid (Table 1 ).
Quantification of phenolic composition in HCFP by HPLC
Discussion This study was based on the extensive use of industrial HCFPs as a dietary supplement in Thailand without scientific testing on their biological properties. Therefore, we aimed at investigating the anti-inflammatory activity of a commercialized fermented broth of H. cordata both in vitro (LPS-induced RAW264.7 model) and in vivo (carrageenan-induced paw edema model). To study the anti-inflammatory potential of the industrial HCFP, the inherent cytotoxic effects of the HCFP extracts on the cellular model used in this study were predetermined using assay. All concentrations of the HCFP extracts used in this study induced negligible cytotoxic effects on RAW264.7 macrophages (cell viability > 90%) (Fig 1A and 1B ), indicating that the inhibitory effect of both extracts on production of inflammatory mediators is not attributed to cytotoxicity. LPS from gram-negative bacteria has been shown to possess a dose-dependent cytotoxic activity in RAW264.7 cells , therefore, the non-toxic concentration of LPS was predetermined (Data not shown). LPS at concentration of 1 μg/ml was used in the present study, and not toxic to RAW264.7 cells, consistent with the result from previous study . In the present study, we reported for the first time that the industrial HCFP possessed antiinflammatory activity. Both HCFP aqueous and methanolic extracts successfully inhibited the production of inflammatory mediators (NO and PGE 2 ). During the inflammatory process, large amounts of the pro-inflammatory mediators like nitric oxide and prostaglandins E 2 are generated by the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), respectively. Nitric oxide is one of pro-inflammatory mediator that responds to pathogenic infections. During the inflammatory process, NO is generated by macrophages to eliminate foreign pathogens, recruiting other cells to the infected area and subsequently resolving the inflammation. However, the excessive amount of NO is also harmful to normal tissue surrounding the infected area because it binds with other superoxides radical and acts as a reactive radical to damage normal cell function. Gram negative bacterial LPS is well known to increase iNOS expression and NO production, leading to the initiation of an inflammatory response . Therefore, the inhibition of NO production is a key therapeutic consideration in both searching for anti-inflammatory agents and developing a novel treatment for inflammatory disorders. In the present study, both aqueous and methanolic extracts of H. cordata fermentation product reduced NO production in LPS-stimulated RAW264.7 cells in a concentration dependent manner (Fig 1C and 1D ). The decreased NO production was correlated well with the dose-dependent decrease of iNOS mRNA ( TNF-α, IL-1β, and IL-6 are the main pro-inflammatory cytokines that are primarily produced by macrophages and have various pro-inflammatory effects on many cell types [31, 32] . Over-production of TNF-α caused the release of various inflammatory mediators including NO, PGE 2 , IL-1β and IL-6. Excessive production of cytokines (TNF-α, IL-1β and IL-6) has linked in several physiological effects, including septic shock, inflammation and cytotoxicity . Thus, the inhibition of cytokine production or function is a key mechanism in the control of inflammation . In the present study, aqueous and methanolic extracts of H. cordata fermentation product reduced production of cytokines in LPS-stimulated RAW264.7 cells in a concentration-dependent manner both at mRNA ( Carrageenan-induced paw edema is an animal model suitable for evaluating inhibition of edema. Biphasic edema induced by carrageenan , includes the first phase (1 h) involving the release of serotonin and histamine and the second phase (over 1 h) mediating by prostaglandins, cyclooxygenase products. In the present study, both doses of the industrial HCFP significantly reduced paw edema at 2 and 3 h after carrageenan injection. This finding suggests that HCFP produces an anti-edematous effect during the second phase which is similar to DCF (Fig 5 ). Interestingly, the highest dose of HCFP (6.16 mL/kg) showed a significant reduction of paw edema at 1 h, suggesting an anti-edematous effect during the first phase. Further animal study on the mechanism underlying inhibition of the first/second phase edema is of interest. The identification of active components in HCFP extracts is an important pharmacological goal. Our HPLC results demonstrated that the amount of all identified phenolic acids in HCFP methanolic extract were much greater than those in HCFP aqueous extract (Table 1 ). Syringic acid was present in the greatest amounts in both HCFP aqueous (88.23 μg/g of extract) and methanolic (2,268.34 μg/g of extract) extracts, followed by vanillic, p-hydroxybenzoic, and ferulic acids, respectively (Fig 6 and Table 1 ). Yoo et al. demonstrated that syringic, vanillic, p-hydroxybenzoic and ferulic acids. In addition, p-coumaric acid has been shown to possess anti-inflammatory activity both in vitro and in vivo [37, 38] . Accordingly, these phenolic acids may contribute to HCFP-mediated inhibition of the production of inflammatory cytokines and mediators. Phenolic acids in the water-soluble constituents of H. cordata fermentation product were previously identified and quantified , but their amounts were greater than those found in the present HCFP aqueous extract (Table 1 ). The discrepancy may be due to a lot-to-lot variation of the industrial HCFP. Further study on synergistic anti-inflammatory effects of HCFP individual phenolic acids both in vitro and in vivo is of interest.
Conclusions Our results demonstrated that the aqueous and methanolic extracts of H. cordata fermentation product possessed anti-inflammatory activity by inhibiting the production of NO, PGE 2 and inflammatory cytokines (TNF-α, IL-1β, IL-6) in LPS-stimulated RAW264.7 cells. The antiinflammatory activity of the industrial HCFP was confirmed by the inhibition of inflammation in the carrageenan-induced rat paw edema model. Our results suggest that this industrial HCFP may be considered as an anti-inflammatory dietary supplement. The health benefits of this industrial HCFP warrant further clinical studies. Fig 1 . 1 Fig 1. Effect of aqueous and methanolic extracts of HCFP on RAW264.7 cell viability (A, B), NO production (C, D) and PGE 2 levels (E, F) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were incubated with aqueous (5-1,500 μg/mL) and methanolic (4-18 μg/mL) extracts for 24 h. Cell viability was assessed by MTT assay. The results were reported as a percentage of cell viability compared with untreated controls and expressed as mean ± S.D. of three independent experiments. For determinations of NO production and PGE 2 levels in LPS-stimulated RAW264.7 cells, the cells were pre-treated with indicated concentrations of aqueous (C, E) and methanolic (D, F) extracts for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. The nitrite production and PGE 2 levels in cultured medium were determined by using Griess reagent and PGE 2 EIA kit, respectively. Statistically significant inhibition of NO production and reduction of PGE 2 levels ( p < 0.05) were found as compared with the LPS group. Data were obtained from three and two independent experiments, respectively.
Fig 2) or protein (Fig 3) levels of COX-2. Similarly, COX-2 mRNA induction was not significantly suppressed by pre-incubation with both HCFP aqueous (50-750 μg/mL) (Fig 2A and 2B) and methanolic (8-12 μg/mL) (Fig 2C and 2D) extracts. COX-2 protein level was not significantly decreased for pre-incubation with both HCFP aqueous (50-750 μg/mL) (Fig 3A and 3C) and methanolic (4-12 μg/mL) (Fig 3B and 3D) extracts. The above results suggest that inhibition of NO and PGE 2 production
Fig 2 . 2 Fig 2. Effect of aqueous and methanolic extracts of HCFP on mRNA expression of iNOS, COX-2, IL-1β, TNF-α, and IL-6 in LPS-stimulated RAW264.7 cells. The cells were pre-treated with indicated concentrations of aqueous and methanolic extracts for 2 h and then stimulated with LPS (1 μg/mL) for 6 h. The mRNA expression levels of aqueous extract-treated (A) and methanolic extract-treated (C) cells were determined by reverse transcription-PCR. Bar graphs showed the relative fold of mRNA expression of aqueous extract-treated (B) and methanolic extract-treated (D) cells, p < 0.05 compared with the LPS group. https://doi.org/10.1371/journal.pone.0230645.g002
Fig 3 . 3 Fig 3. Effect of aqueous (A) and methanolic (B) extracts of HCFP on protein levels of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. The cells were pretreated with the indicated concentration of aqueous extract or phenolic extract for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. The protein expression levels were analyzed by western blot. Bar graphs showed the relative fold of protein expression in aqueous-(C) and methanolic-(D) treated cells. Data were expressed as mean ± S.D. p < 0.05 compared with the LPS group. https://doi.org/10.1371/journal.pone.0230645.g003
Fig 4 . 4 Fig 4. Effect of HCFP on production of IL-1β (A, B), IL-6 (C, D) and TNF-α (E, F) in LPS-stimulated RAW264.7 cells. The cells were pretreated with the indicated concentration of aqueous extract or phenolic extract for 2 h then stimulated with LPS (1 μg/mL) for 24 h. The IL-1β, IL-6, and TNF-α in cultured medium were determined by ELISA kits. Data were expressed as the mean±S.D. of two independent experiments. Statistically significant inhibitions of IL-1β, IL-6 and TNF-α production ( p < 0.05) were found as compared with the LPS group. https://doi.org/10.1371/journal.pone.0230645.g004
The component profiles of HCFP aqueous and methanolic extracts were analyzed by HPLC. The representative chromatograms were shown in Fig 6. Six phenolic acids were identified in HCFP aqueous extract including p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and gallic acids (Fig 6B and Table
Fig 5 . 5 Fig 5. Effect of HCFP and DCF on carrageenan-induced paw edema in Wistar rats. Bar graphs show percentages of changes in paw edema. Data are expressed as mean ± SD of n = 6 rats/group. Asterisk " " indicates a significant difference at p < 0.05 as compared with the control group. https://doi.org/10.1371/journal.pone.0230645.g005
Fig 6 . 6 Fig 6. HPLC chromatograms of phenolic acid standards (A, C) and base hydrolyzed HCFP aqueous (B) and methanolic (D) extracts, where 1 = gallic acid, 2 = protocatechuic acid, 3 = p-hydroxybenzoic acid, 4 = vanillic acid, 5 = caffeic acid, 6 = m-hydroxybenzaldehyde, 7 = syringic acid, 8 = p-coumaric acid, 9 = ferulic acid and 10 = sinapinic acid. The m-hydroxybenzaldehyde was used as an internal standard (I.S.). https://doi.org/10.1371/journal.pone.0230645.g006
Fig 2 ) 2 and protein (Fig 3) levels. PGE 2 , an inflammatory mediator, is produced by the metabolism of arachidonic acid by COX enzymes at inflammatory sites . PGE 2 production increased following LPS treatment. After the RAW264.7 cells were pre-treated with HCFP aqueous and methanolic extracts, the PGE 2 levels in LPS-stimulated RAW264.7 cells were decreased in a dose-dependent fashion (Fig 1E and 1F). However, the NSAID diclofenac (25 μg/mL) exhibited more potent inhibitory activity against PGE 2 production than HCFP extracts at all concentrations tested. The decreased PGE 2 production (Fig 1E and 1F) in both DCF and HCFP treatments was not correlated well with the COX-2 mRNA (Fig 2) and protein (Fig 3) levels. The reduced PGE 2 levels may be due to the inhibition of COX-2 activity by DCF and HCFP treatments. Indeed, DCF has been shown to selectively inhibit COX-2 activity .
Table 1 . 1 Phenolic acid compositions of aqueous and methanolic extracts of HCFP. a Results are expressed as means ± SD of three determinations. n.d., not detected. https://doi.org/10.1371/journal.pone.0230645.t001
Fig 2 ) 2 and protein (Fig 4) levels.
PLOS ONE | https://doi.org/10.1371/journal.pone.0230645March 25, 2020
PLOS ONE | https://doi.org/10.1371/journal.pone.0230645 March 25, 2020 / 18
|
10.2196/43376
|
cc-by
| null | null |
openalex
|
Background: Chronic musculoskeletal pain (CMSP) affects between 13% and 47% of the population, with a global growth rate of 20.3% within the last 15 years, suggesting that there is a high need for effective treatments. Pain diaries have long been a common tool in nonpharmacological pain treatment for monitoring and providing feedback on patients' symptoms in daily life. More recently, positive refocusing techniques have come to be used, promoting pain-free episodes and positive outcomes rather than focusing on managing the pain. Objective: This study aims to evaluate the feasibility (ie, acceptability, intervention adherence, and fidelity) and initial signals of efficacy of the PerPAIN app, an ecological momentary intervention for patients with CMSP. The app comprises digitalized monitoring using the experience sampling method (ESM) and feedback. In addition, the patients receive 3 microinterventions targeted at refocusing of attention on positive events.
Methods: In a microrandomized trial, we will recruit 35 patients with CMSP who will be offered the app for 12 weeks. Participants will be prompted to fill out 4 ESM monitoring questionnaires a day assessing information on their current context and the proximal outcome variables: absence of pain, positive mood, and subjective activity. Participants will be randomized daily and weekly to receive no feedback, verbal feedback, or visual feedback on proximal outcomes assessed by the ESM. In addition, the app will encourage participants to complete 3 microinterventions based on positive psychology and cognitive behavioral therapy techniques. These microinterventions are prompts to report joyful moments and everyday successes or to plan pleasant activities. AfterIntroduction
Background Musculoskeletal pain is defined as chronic if it exceeds a time frame of 3 months or recurs for >3 months . Chronic musculoskeletal pain (CMSP) is estimated to affect between 13% and 47% of the population worldwide (eg, in the European Union , the United States , Canada , Australia , and low-to middle-income countries [6, 7] ). The global disease burden caused by CMSP has been increasing by 20 .3% in the last 15 years . Therefore, from a public health perspective, there is an urgent need for accessible and effective treatments. To determine the etiology and treatment of chronic pain, it is now widely suggested that a biopsychosocial model be followed [1, 9, 10] . CMSP is associated with a range of symptoms and disorders, mainly comorbid depression and anxiety , as well as low self-esteem , attention to and fear of pain [21, 22] , and avoidance behaviors . Therefore, targeting psychological and behavioral aspects can be effective in treating chronic pain [26, 27] . Diary methods including the experience sampling method (ESM) [28, 29] , where participants are prompted to answer several questionnaires per day during their daily life, have become common for monitoring psychological and somatic symptoms, yielding sufficient response compliance across health conditions and age groups . Pain diaries, specifically paper or electronic versions, have proven high satisfaction, feasibility, and compliance for patients with CMSP, providing good psychometric properties . However, using ESM pain diaries that remind participants to rate their level of pain once or several times per day or provide personalized feedback may hold restrictions, as this may promote attention to pain, that is, hypervigilance or attentional bias , which in turn may even increase pain intensity . This is why it has been suggested to focus on positive psychological processes that may broaden attentional focus to positive aspects in life [43, 44] and away from pain. Similarly, positive interventions may be a strategy to promote a focus on resources and resilience [42, 45, 46] . Previous studies have shown that positive interventions that aim to increase awareness of positive aspects of life can improve pain intensity and catastrophizing, happiness, positive affect, depression, and anxiety . In addition, targeting psychological aspects and comorbidities of chronic pain, cognitive behavioral therapy for low self-esteem has been shown to increase self-esteem, psychological functioning, and depression in populations with transdiagnostic psychiatric disorders [50, 51] . Behavioral activation or activity scheduling aimed at increasing positive activities and experiences has shown a large meta-analytic effect in the treatment of depression and first positive evidence in the treatment of chronic pain [53, 54] . Following recent advances in digital technology, mobile health (mHealth) approaches such as ecological momentary interventions (EMIs) that deliver psychological treatment components in daily life [55, 56] can be a promising way to provide early, low-level, accessible, and scalable treatment [57, 58] . As EMIs are provided via mobile phone apps, they may help overcome barriers that can arise for interventions delivered in the clinical setting [28, 55, 59] . Several studies have shown that app-based approaches are feasible and can be effective in the treatment of chronic pain . Furthermore, when patients with chronic pain are offered digitalized monitoring and feedback, they are willing to accept less frequent physician consultations . Within the PerPAIN consortium investigating whether personalized allocation to psychological treatment approaches can enhance treatment effects in CMSP , we developed the PerPAIN app. The PerPAIN app targets positive refocusing of attention to positive events using digitalized monitoring, feedback, and microinterventions in daily life. Thus, the PerPAIN app is a complex intervention comprising several intervention components delivered at varying time points. To evaluate behavioral interventions that comprise multiple intervention components, Collins et al introduced a new approach, that is, the multiphase optimization strategy (MOST) [65, 66] . In addition to the preparation and evaluation phases of an intervention, MOST introduces an optimization phase in which individual intervention components can be evaluated separately according to predefined optimization criteria [65, 66] . This information can then be used to optimize the intervention as a whole to proceed to the evaluation phase [65, 66] .
Objective In this paper, we describe an optimization trial to evaluate the intervention components of the PerPAIN app in line with MOST [65, 66] . Specifically, we designed a microrandomized trial, an experimental research design developed to evaluate and optimize mHealth interventions (for a comprehensive introduction, see Qian et al ). By repeatedly randomizing individuals to different forms of an intervention component (ie, within-person randomization), this research design allows the investigation of the immediate effects of intervention components on repeatedly measured proximal outcomes , as opposed to distal outcomes measured once at the end of treatment or follow-up. In this microrandomized trial, we aim to (1) establish the feasibility for conducting a microrandomized trial in patients with CMSP (based on acceptability, satisfaction, compliance, adherence, and fidelity of delivering the intervention) and ( 2 ) investigate initial signals of efficacy of the feedback component (ie, daily and weekly feedback presented as texts or graphs) and the exercises delivered in microinterventions (namely, the journal of joyful moments, the positive data log, and the activity planner) on the proximal outcomes. To this end, we hypothesize that feasibility criteria for conducting a microrandomized trial evaluating the PerPAIN app in patients with CMSP will be met (hypothesis 1). Second, we hypothesize that within participants, momentary absence of pain (candidate primary proximal outcome) after providing feedback (as text or graph) at the end of a training day on which participants reported at least medium (ie, >=4) absence of pain, positive mood, or subjective activity will, on average across time, be higher compared with time points when no feedback was provided on such days (within-person control condition; hypothesis 2). Third, we hypothesize that within participants, momentary (a) positive mood and (b) subjective activity (candidate secondary proximal outcomes) after providing feedback (as text or graph) at the end of a training day on which participants reported at least medium (ie, >=4) absence of pain, positive mood, or subjective activity will, on average across time, be higher compared with time points when no feedback was provided on such days (within-person control condition; hypothesis 3a and 3b). Finally, we hypothesize that within participants, momentary (a) absence of pain, (b) positive mood, and (c) subjective activity after delivering a microintervention (ie, journal of joyful moments, positive data log, or daily activity planner) at an individually predefined time point will, on average across time, be higher compared with comparable time points on days on which no microintervention was delivered (within-person control condition; hypothesis 4a, 4b, and 4c). We will also explore differences between the 2 types of feedback presentation and 3 types of microinterventions in terms of efficacy and time trends across the 12-week intervention period. In this paper, we describe the PerPAIN app and its intervention components in detail as well as the microrandomized trial implemented to investigate the feasibility and initial signals of efficacy. For a description of the clinical trial that this microrandomized trial is embedded in, we refer to the protocol by Beiner et al .
Methods
Procedure, Study Design, and Randomization A total of 35 patients with CMSP will be recruited to undergo the PerPAIN mobile training, an mHealth app targeted at refocusing of attention on positive events, comprising digitalized monitoring using the ESM, personalized feedback, and EMIs. During a one-on-one structured briefing session, the participants will receive the study app preinstalled on a study smartphone. A trained member of the study team will explain the basic functionalities of the device and app to the participant and answer any potential questions before the participant takes the device into their daily life. The app sends different types of prompts each day for 12 weeks, including monitoring questionnaires, feedback presentations, and microinterventions. Answering all prompts requires approximately 15 minutes per day. A trained member of the study team checks in with the participants up to 6 times to answer any potential questions. The app follows a microrandomized design to evaluate the intervention components (see Multimedia Appendix 1 for a visualization). The training follows a fixed schedule across 12 weeks and entails 4 types of randomization procedures, resulting in a total of t=143 decision points, that is, time points when automated randomization of an intervention component takes place in the backend of the app. First, every evening during the 12 weeks (84 decision points), participants will receive microrandomized daily feedback (text vs graph vs none). Second, at the end of each training week (12 decision points), the participants receive microrandomized weekly feedback (text vs graph vs none). Third, in the second half of the training, the exercises participants are prompted to do as microinterventions are randomized (journal of joyful moments vs positive data log vs daily activity planner vs none) every day for 6 weeks (42 decision points). Fourth, starting from week 8, the weekly activity planner is randomized (on vs off) each week for the remaining 5 weeks (5 decision points). The ESM monitoring questionnaires prompted 5 times per day to serve as proximal outcome assessments. This microrandomized trial is nested in and carried out as part of the PerPAIN trial (see the protocol by Beiner et al for a detailed description of this study, adhering to the Standard Protocol Items: Recommendations for Interventional Trials 2013 checklist ).
Participants For this microrandomized trial, we aim to recruit adult patients (aged >=18 years) experiencing CMSP for >3 months as a symptom of nonspecific chronic back pain, osteoarthritis, fibromyalgia syndrome, or rheumatoid arthritis. Participants must be able to see and use a mobile phone (including visual XSL FO RenderX aids). Exclusion criteria include insufficient or unclear treatment of the underlying disease; severe visual impairment or inability to use a mobile phone; application for retirement or pension pending; ongoing psychotherapy; and severe physical, psychiatric, or neurological comorbidity (see the protocol by Beiner et al for more details). Participants will be recruited primarily via an outpatient pain clinic.
PerPAIN App
Overview The PerPAIN app is a stand-alone, app-based, self-help training with a total duration of 12 weeks, entailing 3 core components. First, it uses the ESM to monitor participants' momentary absence of pain, positive mood, subjective activity, and context several times a day in a positive pain diary (ie, monitoring). Second, the app provides personalized feedback to the participants on the basis of the monitoring data. Third, short exercises based on the principles of cognitive behavioral psychotherapy and positive psychology are provided, which are both taught and implemented within the daily life context (ie, microinterventions). Among these components, gamification elements are used to potentially increase compliance and user engagement.
Monitoring and Feedback The ESM will be used to collect entries for a positive pain diary. The app will prompt participants to answer 4 monitoring questionnaires per day scheduled at semirandom time points within set blocks of time during their waking hours to record their momentary experiences and context. The ESM variables, that is, sleep quality, trust in the intervention, absence of pain, positive mood, and subjective activity, as well as appraisals of momentary and daily context, are rated on a 7-point scale. The monitoring questionnaire consists of 16 to 21 items (depending on the type of prompt and branching rules; see Multimedia Appendix 2 for a full list of items). The PerPAIN app uses the monitoring data to provide feedback on the three main ESM monitoring and outcome variables: (1) absence of pain, (2) positive mood, and (3) subjective activity. We implemented three types of feedback: (1) immediate feedback presented directly after a monitoring questionnaire, (2) daily feedback presented after the last monitoring questionnaire of the day, and (3) weekly feedback presented after each week of monitoring. In accordance with our aim of refocusing of attention on positive events, immediate and daily feedback is only provided if it qualifies as positive. Therefore, immediate and daily feedback on each variable is only displayed if the (mean) score exceeds a predefined threshold of 4, that is, the center of the 7-point scale. Immediate feedback is designed as a type of gamification. After the participant completes filling out the monitoring questionnaire, the conditions for providing positive feedback are checked. Whenever a variable is >=4, meaning the participant is considered to be pain-free, in a good mood, or active, feedback is displayed at the end of the questionnaire, that is, the participant collects a pain-free, feel-good, or activity point, respectively (Multimedia Appendix 3). Daily feedback is made available to the participant in a separate prompt after the last random monitoring questionnaire of the day. Before triggering a prompt for daily feedback, a server checks the conditions for providing positive feedback. Whenever the daily mean score of at least 1 of the 3 main ESM variables is >=4, daily feedback is generated. If, at the same time, one of the other mean scores is <4, feedback for this variable is masked out. For example, if the mean scores of positive mood and subjective activity are both 5 and the mean score of absence of pain is 2, daily feedback is generated, but no value is presented for the absence of pain. Whenever daily feedback is available, a stratified randomization procedure is applied to determine the type of feedback presentation (1:1:1). Feedback is either presented as a bar chart or as a short text, or, in a control condition, no daily feedback is provided. Immediately after the feedback prompt, a monitoring questionnaire containing only the questions regarding the main ESM outcome variables is presented to assess proximal, that is, immediate effects of the daily feedback component. Weekly feedback is made available to the participant in a separate notification after each week of monitoring, that is, in the morning of every 7th day at the earliest possible time for the app to interact with the participant. Unlike daily feedback, weekly feedback is always provided without any restrictions concerning the mean scores of the variables, that is, values <4 are also displayed, as this matches most accurately a paper-pencil positive pain diary that was piloted before this study. Again, a stratified randomization procedure is applied to set the type of feedback presentation (1:1:1). Weekly feedback is either presented as a bar chart or a short text (Multimedia Appendix 3), or, in a control condition, no weekly feedback is provided. Immediately after the feedback prompt, a monitoring questionnaire containing only the questions regarding the main ESM outcome variables is presented to assess the proximal effects of the weekly feedback component.
Microinterventions In addition to the monitoring and feedback components, the PerPAIN app incorporates the principles of EMI. Specifically, it entails 3 exercises implemented as microinterventions alternating across the 12-week training period (Figure 1 ). In the first half of the training, participants are prompted to complete 1 exercise presented as a microintervention per week. As previously used in an EMI targeted at ecological translation of principles of compassion-focused therapy to daily life , the app will offer enhancing, consolidating, and interactive microinterventions. Initially, each exercise is introduced to the participant in an introduction block at the beginning of a new week (ie, enhancing). Once familiar with the microintervention, the participants set the time they wish to be prompted to complete the exercise once a day in a separate notification (ie, consolidation). In addition, if indicated by the participants' answers on a monitoring questionnaire, an exercise is prompted interactively. In the second half of the training, starting from week 7, consolidating and interactive microinterventions alternate each day according to the randomization procedure described in the Procedure, Study Design, and Randomization section. The journal of joyful moments is based on a positive intervention called the "3 good things" intervention . In a continuously growing list structured like a diary, participants collect moments that were happy or joyful for them; things that made them smile; or, in general, made them feel good. The aim of this technique is to draw attention to positive things that happen in daily life, increase well-being, and decrease feelings of depression . Participants can make a new entry ("Please note your joyful moment") once per day at the consolidation prompt, manually whenever they wish to make an entry, or during a monitoring prompt whenever they are likely to have experienced a joyful moment, according to their answers in a monitoring questionnaire. For this purpose, the following conditions were defined: (1) the participant indicates that they are in a good mood, that is, >=4; (2) the participant indicates that their absence of pain is high, that is, >=4; and (3) the participant indicates a high level of activity, that is, >=4 (Multimedia Appendix 4). Once the enhancing exercise is completed, the participant can (in all following training weeks) make an entry in the journal and look at the growing list of joyful moments from the home screen of the app (Multimedia Appendix 5). The positive data log is based on an intervention manual developed to increase self-esteem . The aim of a positive data log is to help correct information-processing errors . The microintervention consists of two parts: (1) collecting small successes during daily life and (2) inferring positive qualities of oneself from the list of successes. When this microintervention is first introduced in week 2, participants collect small or large successes they have experienced in their daily life in a diary. A success can be anything positive or successful the participant does, everything they do well, and anything that they made progress with or put effort into. The aim of this technique is to draw attention to one's own small or big successes in daily life to build self-efficacy and confidence instead of focusing on things that go wrong . Participants can make a new entry ("Please note your success") (1) once per day at the consolidation prompt, (2) manually whenever they wish to make an entry, or (3) during a monitoring prompt whenever they are likely to have had a success according to their answers in a monitoring questionnaire or whenever they do not feel well and will likely benefit from redirecting their attention to something positive. For this purpose, the following conditions were defined: (1) the participant indicates that they have experienced a positive event, (2) the participant indicates that they are at work, school, or university to uncover work-, school-, or university-related successes, and (3) the participant indicates that they are in a bad mood or in pain (Multimedia Appendix 4). In week 5 of the training, when this microintervention is repeated (Figure 1 ), the section on positive qualities is added to the positive data log. After collecting several successes in their daily life, that is, observable behavior, participants are guided to infer positive qualities that underlie those successful behaviors by answering the question "What qualities does a person have that shows these behaviors?" Thus, participants start a second list with their own positive qualities. Each time the participant makes an entry into their positive data log, they are invited to add a positive quality. Once the enhancing of each part of the exercise is completed, the participant can (in all following training weeks) make an entry to the positive data log and look at the growing list of successes and positive qualities from the home screen of the app (Multimedia Appendix 5). The activity planner is based on a manual for behavioral activation , a behavioral therapy technique used in the treatment of depression and chronic pain . In behavioral activation, pleasant activities are systematically increased to change corresponding cognitions. In an introductory block, the participant is educated about the interdependence between pleasant activities and positive thoughts and feelings (Multimedia Appendix 5). The participant then creates their own activity plan for the week in a guided process. From a list of activities from different life domains, that is, sociability, hobbies and free time activities, sports and physical activities, culture and education, nature and gardening, and self-care and delight, the participant is invited to choose 3 activities or insert activities they would like to do in the following week. After choosing each activity, the participant is instructed to define the number of times they would like to do the activity. This step is based on the idea that progress toward specific goals can be monitored easily and can therefore be more motivating . During the activity week, the participant can monitor their progress with their planned activities. Each morning, the participant receives a notification reminding them of their planned activities. Optionally, they can plan an additional activity for the upcoming day only (ie, daily activity planner). Participants can make a new entry ("Please tick off all activities you have done today") once per day at the consolidation prompt or manually by entering the activity planner from the home screen of the app. On the last consolidation prompt of the activity week, the planned and actual frequencies of each activity are presented to the participant, and they can indicate whether they have achieved, exceeded, or not accomplished their goal. The achievement or exceedance of goals is rewarded using the gamification elements described in the Gamification section.
Gamification Gamification is a concept that integrates game elements into nongame contexts. It builds on the engaging and motivating potential of games . In mHealth, gamification elements can be used to increase compliance and shape and reward desired behaviors . After downloading an mHealth app, user engagement can vary greatly (eg, see the study by Rahman et al ), which is why it is a common objective of researchers to design apps to optimally support engagement . In the PerPAIN app, we implemented three gamification elements aimed at increasing compliance and providing rewards: (1) an overall training score, (2) immediate feedback designed as points, and (3) a progress bar. The overall training score increases whenever the participant answers a prompt or makes an entry (Multimedia Appendix 3). Hence, this gamification element is aimed at increasing users' overall engagement with the app. In the overall introduction to the app as well as the enhancing blocks introducing the microinterventions, the participant is informed which actions lead to an increase in the training score (eg, answering a monitoring prompt, making an entry in the journal of joyful moments, or achieving an activity goal). After a monitoring prompt, the participant can receive 3 types of points (ie, pain-free, feel-good, and activity points), whenever the values of the 3 scales were >=4. These points are collected throughout the training and mainly serve as positive feedback but may also reinforce behaviors that lead to reporting higher values of absence of pain, positive mood, or subjective activity in the monitoring questionnaire. The overall user advancement is presented to the participant on a summary page that includes a progress bar displaying progression throughout the training using an icon of a person walking on a path of 12 tiles representing the 12 training weeks. This gamification element aims to increase the transparency and completion rates of the training.
Measures We will assess the feasibility of delivering the smartphone training and the effects of intervention components.
Feasibility Measures In line with previous research on the feasibility of delivering EMIs [70, 81, 82] , feasibility measures will include acceptability, intervention adherence, and fidelity. Acceptability will be assessed in a debriefing questionnaire rating satisfaction with the intervention, ease of use, accessibility, and comprehensiveness [70, 81, 82] . In addition, we will use the user version of the mobile app rating scale to assess the subjective quality of the app. Adherence to the mobile training will be operationalized by the completion rates of the intervention components (ie, mean number of monitoring questionnaires, consolidating or interactive tasks completed/wk, and [mean] number of feedback prompts answered). As a core feature of the PerPAIN training is the delivery of intervention components in daily life, that is, in varying contexts, aiming to support a positive attention focus while reducing transfer effects, we will measure fidelity by the variety of contexts in which participants engage with the app and the proportion of participants who still use the app at the end of the 12-week training period. To establish levels of feasibility, we will classify our feasibility outcomes using a traffic light system into 3 categories, that is, green=feasibility fully established, yellow=feasibility established with a need for modification of the study procedures, and red=feasibility not established (Table 1 ).
Fidelity Adherence Acceptability Category Poor use of the app overall (>=60% of the participants using the app for <3 wk) and poor variability of intervention contexts (>=60% of the participants using the app in 1 context exclusively, as measured by the "current activity" item) Poor compliance to the monitoring and feedback component (>=60% of the participants answering on average <50% of the questionnaires/d and >=60% of the participants viewing their provided feedback on average less than once/wk) and EMI b tasks (ie, mean number of consolidating or interactive EMI tasks completed/wk is <0.8) Low satisfaction in the debriefing questionnaire (ie, mean satisfaction rating of <=3 on a 7-point scale) and the uMARS a (ie, mean subjective quality rating of <=2 on a 5-point scale) Red (feasibility not established) Fair use of the app overall (>=60% of the participants using the app for >=3 wk) and fair variability of intervention contexts (>=60% of the participants using the app in at least 2 contexts, as measured by the "current activity" item) Fair compliance to the monitoring and feedback component (>=60% of the participants answering on average at least 50% of the questionnaires and >=60% of the participants viewing their provided feedback on average at least once/wk) and EMI tasks (ie, mean number of consolidating or interactive EMI tasks completed/wk is >=0.8) Fair satisfaction in the debriefing questionnaire (ie, mean satisfaction rating of >3 on a 7-point scale) and the uMARS (ie, mean subjective quality rating of >2 on a 5-point scale) Yellow (feasibility established but study procedures need modifying) Moderate to high use of the app overall (>=80% of the participants using the app for >= 6 wk) and moderate to high variability of intervention contexts (>=80% of the participants using the app in at least 2 contexts, as measured by the "current activity" item) Moderate to strong compliance to the monitoring and feedback component (>=80% of the participants answering at least 50% of the questionnaires and >=80% of the participants viewing their provided feedback on average at least once/wk) and EMI tasks (ie, mean number of consolidating or interactive EMI tasks completed/wk >=1) Moderate to high satisfaction in the debriefing questionnaire (ie, mean satisfaction rating of >4 on a 7-point scale) and the uMARS (ie, mean subjective quality rating of >3 on a 5-point scale) Green (feasibility fully established) a uMARS: user version of the mobile application rating scale. b EMI: ecological momentary intervention.
Candidate Primary Outcome The candidate primary outcome of the microrandomized trial is the proximal measure of the absence of pain assessed in the monitoring questionnaires. Specifically, we will investigate the immediate effect of the daily feedback component on the absence of pain rating measured on a 7-point scale in the monitoring questionnaire presented either directly after daily feedback was displayed or in a separate prompt without feedback being displayed (control condition), depending on the microrandomization (Multimedia Appendix 1).
Candidate Secondary Outcomes The 2 candidate secondary outcomes of the microrandomized trial are the proximal measures of positive mood and subjective activity assessed on a 7-point scale in the monitoring questionnaires. As for the candidate primary outcome, we will use the ratings given in the monitoring questionnaire presented either directly after daily feedback was displayed or in a separate prompt without feedback being displayed (control condition), depending on the microrandomization.
Time-Fixed Covariates In line with the PerPAIN study , we will use the treatment condition (personalized vs nonpersonalized treatment allocation), gender, and baseline pain severity (as measured by the German version of the Multidimensional Pain Inventory ) as covariates in all models.
Sample Size This microrandomized trial forms part of the PerPAIN randomized controlled trial . Given that we expect to include 35 participants in this microrandomized trial, we performed power analysis for microrandomized trials to determine the effect sizes we will be able to detect in the given study setup with sufficient power of at least 80% testing at α=.05 (for a description of the tool, see Seewald et al ). Regarding the daily feedback component with 1 decision time point for a total of 84 days, we will be able to detect an average proximal treatment effect of 0.16 with a power of 80.4% and a sample size of 35, assuming that at least 1 ESM variable is >4 (therefore, available for feedback) on every second day of the study (ie, availability parameter of 0.5). Regarding the microinterventions with 1 decision time point for a total of 42 days, we will be able to detect an average proximal treatment effect of 0.18 with a power of 80.8%, a sample size of 35, and an availability parameter of 1, as microinterventions will be prompted every day without any restrictions. Intervention components randomized weekly (weekly feedback and weekly activity planner) do not produce sufficient power to be tested for proximal treatment effects in this study.
Assessment of Safety in the Microrandomized Trial Throughout participation in this microrandomized trial, we will monitor any serious adverse events, such as serious incidents resulting in death or hospitalization, and adverse events (see the protocol by Beiner et al for more details). Furthermore, we will record any potential adverse device effects and technical problems reported by participants, such as crashing of the app or bugs. Whenever possible, we will fix technical problems immediately and introduce a new version of the app for future participants using the app.
Statistical Analysis To address hypothesis 1, we will use descriptive statistics and 95% CIs as appropriate to evaluate the feasibility of delivering the PerPAIN training in line with the predefined feasibility criteria described in Table 1 . To test initial signals of efficacy on candidate primary and secondary proximal outcomes, we will use the weighted and centered least squares estimator to estimate the causal excursion effect of the daily feedback component [87, 88] . To investigate whether there is an overall effect (ie, on average across time) of daily feedback on our candidate primary proximal outcome momentary absence of pain (hypothesis 2), we will calculate the weighted and centered least squares estimator by specifying a generalized estimation equation with absence of pain in the subsequent monitoring questionnaire as the proximal outcome, with a treatment indicator specifying whether daily feedback was delivered (=1) or not (=0), including 84 decision points, that is, once every day for 12 weeks. As feedback is only delivered whenever the daily average of one of the main outcome variables was >4, we will create a new variable good_day (1=at least 1 outcome is >4 and 0=no outcome is >4) serving as an availability indicator in the equation. The analysis will be controlled for time-fixed covariates. To address hypotheses 3a and 3b, we will repeat this procedure using our candidate secondary proximal outcomes: (a) momentary positive mood and (b) momentary subjective activity at the subsequent monitoring questionnaire as the proximal outcome. In the next step, to explore the effect of different feedback types individually (ie, as text or graph), we will include 1 treatment indicator for each type of feedback. Furthermore, we will explore whether there is a time trend in the effects of daily feedback by including a variable training_day counting the days in the study (with the value 0 on the first day). To investigate the immediate effects of the microinterventions, we will use momentary absence of pain, positive mood, and subjective activity rated on a 7-point scale in the next monitoring questionnaire prompted after the predefined consolidation time, unless the consolidation prompt was the last prompt of the day, to avoid overnight effects. To determine whether there is an overall effect, that is, on average across time, of the microinterventions (hypotheses 4a, 4b, and 4c), we will specify 3 generalized estimation equations with a treatment indicator specifying whether a microintervention was delivered at the individually predefined consolidation time on that day (=1) or not (=0), that is, control condition, using each outcome: (a) momentary absence of pain, (b) momentary positive mood, and (c) momentary subjective activity at the subsequent prompt as a proximal outcome, including 42 treatment occasions, that is, once every day between week 7 and 12. There will be no availability indicator, and time-fixed covariates will be included. We will then include 1 treatment indicator for each type of intervention to investigate the effects of the 3 microinterventions individually (ie, journal of joyful moments, positive data log, and daily activity planner) and explore the time trend in the effects of the microinterventions by including the training_day indicator. Data analysts will be blinded to the treatment indicators.
Results The Ethics Committee II of the University of Heidelberg approved this study (2020-579N) on August 4, 2020. At the time of submission of this protocol (October 10, 2022), we are working with trial protocol version 6 (October 6, 2021). The outcome assessments are ongoing. The first enrollment in this microrandomized trial was conducted in November 2021. As of October 10, 2022 (the date of submission), 24 participants were enrolled in the microrandomized trial, and 13 participants completed the training. Data will not be accessed for interim analysis before the completion of all outcome assessments. The last assessment for the last participant is scheduled for July 2023, and we expect the results to be published in early 2024.
Discussion Extending the widely used method of symptom monitoring using pain diaries, we will investigate the feasibility and initial signals of efficacy of a newly developed self-help app targeted at refocusing of attention to positive events with different intervention components based on principles of EMI. EMIs offer the great advantage of delivering intervention components in various contexts in daily life [28, . EMIs are frequently implemented as hybrid interventions [70, 81, 82] or stand-alone interventions, such as the PerPAIN app. Owing to the microrandomized design, we will be able to evaluate individual intervention components separately to optimize the intervention as a whole and proceed to the evaluation phase in line with MOST [65, 66] . The results of this optimization trial will show whether delivering an app-based self-help training with different intervention components is feasible. Furthermore, we will investigate its acceptability, satisfaction, compliance, adherence, and fidelity in a population of patients with CMSP. The results of this trial promise first insights into the potential proximal effects of delivering daily feedback and prompting microinterventions to improve the absence of pain and increase positive mood and subjective activity. The results of this trial may suggest ways to optimize these intervention components. For instance, the results may point toward 1 type of feedback presentation (eg, as a graph) or microintervention (eg, activity planner) having greater initial effects than another. Alternatively, there may be initial evidence of a time trend across the 12 weeks of the training, which could inform the setup of future studies. If this trial is successful, the next step will be to proceed to a confirmatory trial with a larger sample powered to detect the expected effect sizes established in this trial. However, for the interpretation of the results of this study, it should be considered that the effect sizes that will be established may be diminished because of the design of the PerPAIN study this microrandomized trial is nested in. As a result of the randomization procedure to investigate a personalized treatment approach (see the protocol by Beiner et al ), 50% (17/35) of the participants in this microrandomized trial will be allocated to participate in the PerPAIN training according to a personalized treatment allocation procedure, whereas the other half will be allocated to the PerPAIN training against the personalization approach.
XSL FO
RenderX Positive psychological approaches seem to be promising for the treatment of CMSP [42, . Therefore, investigating how these approaches can be implemented using mHealth technologies is an important step in tackling the global disease burden caused by CMSP. Overall, EMIs using microinterventions offer a great opportunity to provide low-level treatment in the context of daily life, thereby overcoming barriers to interventions delivered in the clinical context 59 ]. After positive scientific evaluation, this type of treatment promises significant public health impact as it is scalable to a large population of patients with CMSP, which may ensure continuity of care and reduce on-site consultations . Figure 1 . 1 Figure 1. Timeline of the PerPAIN training intervention components.
Table 1 . 1 Feasibility criteria of delivering the PerPAIN training.
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 2 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 3 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 4 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 5 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 6 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 7 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 8 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 9 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 10 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 11 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 12 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 13 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
JMIR Res Protoc 2023 | vol. 12 | e43376 | p. 14 https://www.researchprotocols.org/2023/1/e43376 (page number not for citation purposes)
|
10.21496/ams.2020.002
|
cc-by
| null | null |
openalex
|
The article introduces an innovative concept of a screw of an extruder used for granulation of polymer materials. Three construction solutions were introduced, differing in the construction parameters of the screw. The scope of the research included the numerical strength-thermal analysis of the introduced construction solutions of the screw. The analysis was conducted using the finite element method. Numerical calculations were a thermally coupled FEM analysis, posing a geometrically and thermally nonlinear problem. The obtained results confirmed the sufficient strength of the structure of the screw, subjected to both the pressure caused by the transport of polymer granules as well as the temperature generated by the friction of the granules on the surface of the screw. The numerical tool applied in the research was the commercial program ABAQUS.Introduction An important aspect of developing modern extrusion presses for production of elements made of polymer materials is a proper choice of the geometrical parameters of the press, which allows one to obtain the optimal parameters of the manufacturing process. Many examples describing the influence of the feeding zone on the efficiency and pressure of the material in the plasticizing system of the press1,2 can be found in specialist literature. In this respect the most effective proved to be the extruders with a grooved feed section, which allowed one to obtain a tens of percent increase of effectiveness and pressure 3÷5. Moreover, the quality of the obtained extrudate can be assessed based on the level of mechanical and thermal homogenization of the material, in many cases requiring the use of additional devices, such as static mixers in the process line6. It is especially important in the case of filled materials, among others with nanofillers, which tend to agglomerate, therefore decreasing the quality of the obtained products. According to Głogowska and Sikora7 the modification of polymers by a wide variety of fillers causes numerous changes in processing, mechanical properties and morphology of product structure. Moreover, they confirmed that those properties depend on the proper mixing. The main construction element of the extruder, significantly influencing the effectiveness of mixing the material and operation of the extruder is, among others, the screw. A proper selection of the geometrical parameters of the screw, both in the feeding (cold) zone and the plasticizing (hot) zone has a direct influence on the pressure and temperature distribution in the transported material. The objective of this work is to present innovative concepts of the screw of an extruder with a plasticizing system arranged to cooperate with an innovative rotational barrel segment. The introduced concept is a fully functional solution, examined in terms of strength and temperature, and therefore suitable for the further stages of
Concept and geometrical parameters of the screw of the extruder The subject of the research was a screw of a extruder, adapted for the rotational segment of the barrel. The geometry of the screw was variable, depending on the work zone. Three concepts of the screw with the working length equal 755 mm and diameter equal Ø25 mm were discussed. The concepts differed in terms of the pitch value and winding depth, which led to different pre-stressing of material, respectively: variant I -2.09, variant II -2.43 and variant III -2.29. The construction details of the discussed variants of the screw presented in Fig. 1 also influence the distribution of the reduced stress and the temperature distribution within the screw material.
FEM numerical calculations
Methodology of numerical calculations The numerical calculations were conducted using the finite element method. Numerical simulations consisted of coupled thermal-displacement analysis, a combination of a static strength analysis and a thermal analysis, allowing one to determine the distributions of stress and temperature in the area of the analysed structure 8÷11. In this case, the general formula for the fully-coupled thermal-stress analysis can be presented as follows: Where: Du and Dq are the respective corrections to the incremental displacement and temperature, Kij ( ) 1 are submatrices of the fully coupled Jacobian matrix, and Ru and Rq are the mechanical and thermal residual vectors, respectively. Solving this system of equations requires the use of the asymmetric matrix storage and solution scheme. Furthermore, the mechanical and thermal equations must be solved simultaneously. The method provides quadratic convergence when the solution estimate is within the radius of convergence of the algorithm. The numerical calculations were a geometrical and physical nonlinear problem, solved using the Newton-Raphson incremental-iterative method 12÷15. The numerical tool applied was a commercial package of the finite element method -the ABAQUS program.
Structure of the discrete model The discretization of the geometrical model of the screw was conducted using type C3D4T tetragonal solid elements being 4-node elements with the first-order shape function and full integration, allowing one to consider the thermal degree of freedom in the numerical analysis. The applied method of structure discretization led to creating a discrete model consisting of 127 803 finite elements, which resulted in solving a numerical problem with 1 294 472 nonlinear equations of the coupled analysis. The general view of the discrete model is presented in fig. 2 . The screw was manufactured from 40HM grade steel, for which an elasto-plastic material model was determined. The material properties are presented in table 1 and 2 . Elongation at break [%] 10 Thermal property -steel 40HM K K K K R R uu u u u u T T TT T T a ¬ « o 1⁄4 » ' ' ̄1⁄2 3⁄4 ¿ ̄1⁄2 3⁄4 ¿ Linear expansion coefficient [1/K ] 1.2 10 -5 Heat conduction coefficient l [W/mK] 58 Specific heat [J/kgK] 450
Boundary conditions and load of the discrete model The boundary conditions of the screw model are defined by blocking the translational degrees of freedom of nodes located in the journal bearings in a way presented in fig. 3 . The basic mechanical load was the rotational moment Mc= 200 Nm applied at the end of the journal (fig. 3 ). Additional mechanical load was the pressure caused by the transported material, which was imitated by applying pressure to the surface of the screw. The exponential distribution of pressure along the axis of the screw, the maximum value of which was equal pmax = 25 MPa (fig. 4 ) was defined. Thermal load caused by the friction of the transported material in the cold zone of the extruder and the temperature in the hot zone were imitated by applying the temperature fields on the surface of the screw. In order to achieve this the exponential distribution of temperature was defined. Its maximum value occurring in the end of the hot zone was equal Tmax = 190C -fig. 5 . The initial temperature of the numerical model was assumed to be T0 = 22C.
Results of the numerical calculations The strength and stiffness analyses of the individual elements of the structure were conducted on the basis of the reduced stress distributions, determined according to the Huber-Mises-Hencky (H-M-H) strength hypothesis as well as the maps of node dislocations in the area of the analysed structure. The presented results pertain to the case of mechanical and thermal load of the structure of the screw, equal the constant operation of the device, equal 5 hours. Figure 6 shows the maps of the node dislocations of the screw model expressed in [mm].
( ) a ( ) b ( ) c The obtained values of the node dislocation are similar for all three variants and remain in the range 2.11÷2.32mm. Upon referring the obtained results to the dimensions of the screw it can be stated that the structure of the screw remains sufficiently stiff subjected to mechanical and thermal load in the constant operation. Fig. 7 shows the maps of H-MH reduced stress expressed in [MPa], allowing for the assessment of the screw structure strength. Upon analysing the obtained values of the reduced stress it can be stated that in all the examined variants the level of the maximum stress in similar and oscillates around σz ≈ 875÷879MPa. The areas in which the highest gradients of the reduced stress are located are marked in red and occur in the front part (drive application) as well as in the end part of the screw. The obtained values of the reduced stress are high, but do not exceed the value of the yield point. According to the assumed material properties of 40HM grade steel it is equal Re=880 MPa. Therefore, the level of the reduced The temperature distributions in the material of the screw were also analysed. Figure 8 shows maps of the temperature expressed in [oC] for the respective construction variants of the screw. The obtained results allow one to determine the temperature distribution in the area of the structure during constant operation of the extruder.
( ) a ( ) b ( ) c Upon analysing the temperature distributions, it can be observed that in the case of variant I maximum temperature (marked red) occurs on the significant length of the screw, which ensures the optimal granulate plasticizing conditions in the hot zone of the extruder. In the case of variants II and III the area of maximum temperature is significantly shorter, which render those construction solutions less beneficial in the case of the effectiveness of the extruder and optimization of the manufacturing parameters. The direct cause of this phenomenon is the variety of geometrical parameters of the screw windings.
Conclusions The study presents innovative construction solutions for the screw of an extruder, adopted for the rotating segment of the barrel. Three construction variants were proposed, differing in the geometrical parameters of the screw windings. A numerical analysis allowed for assessing the strength and temperature distribution in the screw during constant operation of the press. The thermally coupled analysis allowed for considering all aspects of the structure load during the manufacturing process, considering both strength and thermal aspects. The conducted calculations confirmed the sufficient strength and stiffness of the structure subjected to multi-axis load conditions, which ensures the most stable and safe operation of the extruder. An analysis of the temperature distributions during constant operating of the press allowed for selecting the most construction solution -variant I, ensuring the most optimal working conditions, which directly influences the effectiveness of the manufacturing process. The proposed concept of the screw is an innovative solution for the extruder with so-called active grooved feed section and will be commercialized by manufacturing and implementing the extruder to the manufacturing process. Fig. 1 : 1 Fig. 1: Geometrical model of the screw: a) general view, b) variant I, c) variant II, d) variant III.
Fig. 2 : 2 Fig. 2: Discrete model of the screw.
Fig. 3 : 3 Fig. 3: Definition of the boundary conditions of the screw.
Fig. 4 : 4 Fig. 4: Exponential distribution of pressure on the surface of the screw.
Fig. 5 : 5 Fig. 5: Exponential distribution of temperature on the surface of the screw
Fig. 6 : 6 Fig. 6: Maps of the node dislocation of the screw model: a) variant I, b) variant II, c) variant III.
Fig. 7 : 7 Fig. 7: Maps of the reduced stress H-M-H in the screw structure: a) variant I, b) variant II, c) variant III.
stress occurring in the structure does not threaten the proper work of the structure.
Fig. 8 : 8 Fig. 8: Maps of temperature distribution in the structure of the screw: a) variant I, b) variant II, c) variant III.
Table 1 : 1 Mechanical properties of 40HM grade steel. Mechanical property -steel 40HM Density r [kg/m 3 ] 7860 Young modulus E [Pa] 2.110 5 Poisson Coefficient [ -] 0.3 Yield strength Re [Pa] 8.810 2 Strength limit Rm [Pa] 1.0310 3
Table 2 : 2 Thermal properties of 40HM grade steel.
|
10.48550/arxiv.1705.03274
|
cc-by
| null | null |
openalex
|
After Run 1 of the LHC, global fits to b → s observables show a deviation from the Standard Model (SM) with a significance of ∼ 4 standard devations. An example of a b → s process is the decay of a B 0 s meson into two muons (B 0 s → μ + μ -). The latest analysis of B 0 (s) → μ + μ - decays by LHCb with Run 1 and Run 2 data is presented. The B 0 s → μ + μ -decay is observed for the first time by a single experiment. In addition, the first measurement of the B 0 s → μ + μ - effective lifetime is performed. No significant excess of B 0 → μ + μ -decays is observed. All results are consistent with the SM and constrain New Physics in b → s processes.Introduction In the SM, Flavour Changing Neutral Currents (FCNCs) occur at loop level and are suppressed by the GIM mechanism, and sometimes helicity suppression. As New Physics (NP) is not necessarily suppressed, FCNCs probe physics at energies beyond the LHC centre-of-mass energy. One such FCNC is the b → s transition. Several theory groups have performed global fits to b → s observables in the Effective Field Theory (EFT) framework, and find that the data deviate by ∼ 4 standard deviations with respect to the SM 1, 2, 3 . One example of a b → s transition is the decay of a B 0 s meson into two muons (B 0 s → μ + μ -). For B 0 (s) → μ + μ -decays, the decay amplitude can be written as A(B 0 (s) → μ + μ -) = μ + μ -|H ef f |B 0 (s) = G F √ 2 V * t(d,s) V tb i∈10,S,P C ( ) i μ + μ -|O ( ) i |B 0 (s) , (1) where As B 0 (s) → μ + μ -decays are purely leptonic, the hadronic part of the amplitude reduces to the decay constant, which is well determined using lattice QCD 4 . The branching fractions of B 0 → μ + μ -and B 0 s → μ + μ -are consequently well predicted in the SM 5, 6 : G F is the Fermi constant, V * t(d, B(B 0 s → μ + μ -) = (3.57 ± 0.18) × 10 -9 (2) B(B 0 → μ + μ -) = (1.06 ± 0.06) × 10 -10 (3)
arXiv:1705.03274v1 [hep-ex] 9 May 2017 A second observable in the B 0 (s) → μ + μ -system is the effective lifetime of B 0 s → μ + μ -7 . B 0 (s) mesons mix with their anti-particles and form mass (and lifetime) eigenstates. The masseigenstate rate asymmetry is given by A ∆Γ = Γ(B 0 (s),H → μ + μ -) -Γ(B 0 (s),L → μ + μ -) Γ(B 0 (s),H → μ + μ -) + Γ(B 0 (s),L → μ + μ -) (4) In the SM, A ∆Γ = 1, which means that only the heavy B 0 (s) eigenstate decays to two muons. For B 0 s mesons, the lifetime difference between their two eigenstates is measured to be ∆Γ/Γ = 0.12 a By measuring the effective lifetime from the untagged decay distribution, the mass-eigenstate rate asymmetry is probed. The effective lifetime of B 0 s → μ + μ -is an independent and complementary observable to the branching fraction. Here, the new LHCb analysis of B 0 (s) → μ + μ -observables is presented 8 . It is based on data corresponding to an integrated luminosity of 1 fb -1 of pp collisions at a centre-of-mass energy of √ s = 7 TeV, 2 fb -1 at √ s = 8 TeV and 1.4 fb -1 at √ s = 13 TeV. The first two datasets are referred to as Run 1, the latter as Run 2. In Section 2, the branching fraction measurement is presented. In Section 3, the effective lifetime measurement is presented. As there is a large overlap in selection and analysis strategy, only differences with the branching fraction measurement are mentioned in the latter Section. 2 B 0 s → μ + μ -and B 0 → μ + μ -branching fraction measurement 2.
Backgrounds and selection The analysis strategy is to search for muon pairs with opposite charges, m μ + μ -∈ [4900, 6000] MeV b and a well reconstructed, clearly displaced decay vertex. Four background categories exist for the branching fraction measurement. First, bb → μμX decays, where both muons combine to fake a signal. This is called combinatorial background, and all other backgrounds are hereafter referred to as peaking backgrounds. Second, B 0 (s) → h + h ( )- decays, where h ∈ [K, π] and both hadrons are misidentified as muons. Third, B 0 → π -μ + ν μ , B 0 s → K -μ + ν μ , and Λ 0 b → pμ -ν, where one hadron is misidentified as a muon and the neutrino is not reconstructed. Fourth, B + c → J/ψ(→ μ + μ -)μ + ν μ and B 0(+) → π 0(+) μ + μ -decays, where two real muons are combined and one particle is not reconstructed. To reject both combinatorial and peaking backgrounds, two multivariate classifiers are used. For the separation of B 0 (s) → μ + μ -from combinatorial background, a Boosted Decision Tree (BDT) is trained. This BDT is used to divide the sample based on signal purity. The main discriminating variables in this BDT are based on track isolation, as bb → μμX decays have extra tracks close to the muon tracks. To reject peaking backgrounds, neural networks that discriminate between muons and hadrons are trained. The neural network selection is optimised for the B(B 0 → μ + μ -) measurement, as due to its lower mass it is more affected by peaking backgrounds. Simulation is used for each peaking background to estimate the number of events after selection, as well as the mass and BDT shape. The main backgrounds (B 0 (s) → h + h ( )-, B 0 → π -μ + ν μ , B 0 s → K -μ + ν μ ) are also controlled using a fit to data with the hμ mass hypothesis.
Signal calibration and normalisation To convert a yield after selection into a branching fraction, other B decays with known branching fractions are used for normalisation. In addition, the mass and BDT shape of the signal have a For B 0 mesons, no lifetime difference has been observed. It is expected to be O(10 -3 ). The effective lifetime of the B 0 → μ + μ -decay is not measured due to the small lifetime difference and its small branching fraction. b For reference, the B 0 s mass is 5367 MeV/c 2 and the B 0 mass is 5280 MeV/c 2 . to be calibrated. The normalisation is performed with B + → J/ψ(→ μ + μ -)K + and B 0 → K + π -decays. The two muons in the final state for the B + → J/ψ(→ μ + μ -)K + decay are detected similarly to the muons from B 0 (s) → μ + μ -. The efficiency to detect the extra kaon track in the final state is corrected for using simulation. For B 0 → K + π -, the kinematics are very similar to B 0 (s) → μ + μ -, which means the same BDT can be applied. However, the trigger and PID selection for hadrons and muons are very different, and are corrected for using a data-driven approach. For the BDT calibration, fits to B 0 → K + π -decays per BDT bin are used to determine the B 0 (s) → μ + μ -BDT distributions. The same corrections as for the normalisation are applied, and the BDT distribution is found to be consistent with simulation.
Branching fraction fit and results To measure both branching fractions, a simultaneous maximum likelihood fit for B(B 0 s → μ + μ -) and B(B 0 → μ + μ -) is performed to m μ + μ -in bins of BDT per dataset (Run 1 and Run 2). The only free nuisance parameters are the combinatorial background shape parameters and yields. The shape is the same for all BDT bins in a dataset, the yields are free for each BDT bin. From the fit, the B 0 s → μ + μ -decay is observed with a statistical significance of 7.8 standard deviations, and its branching fraction is measured to be B(B 0 s → μ + μ -) = (3.0 ± 0.6 +0.3 -0.2 ) × 10 -9 , where the first uncertainty is statistical and the second systematic. The statistical uncertainty is obtained by repeating the fit with all nuisance parameters fixed. No significant excess of B 0 → μ + μ -decays is observed, and a 95% confidence level upper limit is determined, B(B 0 → μ + μ -) < 3.4 × 10 -10 .
3 Effective lifetime measurement of B 0 s → μ + μ - In contrast to the branching fraction, the effective lifetime is measured for the B 0 s → μ + μ -decay only. Therefore, the selection and fitting procedure are optimised independently. First, events with m μ + μ -< 5320 MeV, and thus all peaking backgrounds, as well as B 0 → μ + μ - events, are cut away. Second, the PID selection is loosened, as the mass cut makes the measurement less sensitive to peaking backgrounds. Third, a cut is applied on the BDT, instead of using the BDT to divide the sample in bins. With the sPlot method 9 , a mass fit is used to background-subtract the decay time distribution. The mass fit contains just two shapes (B 0 s → μ + μ -and combinatorial background). The background-subtracted decay time distribution is fitted with an exponential, convoluted with an acceptance function. The effective lifetime is measured for the first time and found to be τ (B 0 s → μ + μ -) = 2.04 ± 0.44 ± 0.05 ps. This measurement is consistent with the A ∆Γ = +1(-1) hypothesis at 1.0 (1.4) standard deviations. The current experimental precision does not set a strong constraint, but shows the potential LHCb will have to constrain NP using the effective lifetime.
Conclusions The B 0 s → μ + μ -decay is observed with a significance of 7.8 standard deviations, and its branching fraction is measured to be B(B 0 s → μ + μ -) = (3.0±0.6 +0.3 -0.2 )×10 -9 , where the first uncertainty is statistical and the second systematic. In addition, the first measurement of the B 0 s → μ + μ - effective lifetime is performed: τ (B 0 s → μ + μ -) = 2.04 ± 0.44 ± 0.05 ps. No significant excess of B 0 → μ + μ -decays is observed, and a 95% confidence level upper limit is determined, B(B 0 → μ + μ -) < 3.4×10 -10 . All results are consistent with the SM and constrain New Physics in b → s processes. Already, these results have sparked interesting discussions 6, 10, 11, 12 . iP s) and V tb are CKM elements, C are non-perturbative Wilson operators. These operators describe axial vec-) contributions respectively, and a prime indicates a right-handed contribution. In the SM, only C 10 contributes. Interestingly, contributions from C ( ) 10 are helicity suppressed, while (pseudo-)scalar contributions are not. Therefore, B 0 (s) → μ + μ - observables are very sensitive to (pseudo-)scalar NP.
Figure 2 - 2 Figure 2 -(left) Mass distribution of B 0 s → μ + μ -candidates. The result of the fit is overlaid. (right) Backgroundsubtracted decay-time distribution with the fit result superimposed.
|
10.33102/2022271
|
cc-by
| null | null |
openalex
|
Multiple Sclerosis is an inflammatory demyelinating disease that affects the central nervous system, and is uncommon among the Asian population. It can present with a variety of symptoms, depending on the location of the demyelinating plaques, which could also potentially mimic a range of neurological and non-neurological disorders. We report a 24-year-old woman who presented with recurrent non-specific lower limb numbness, without any other neurological symptoms or signs. The symptoms were presumed to be nonspecific, but they re-occurred eight months later, leading to a more extensive investigation, including whole spine and brain MRI. The MRI showed multiple white matter lesions that are hyperintense on T2W/FLAIR, seen at the right corona radiata, bilateral parietal lobes, right periventricular region and spinal cord, with a simultaneous presence of gadolinium-enhancing and nonenhancing lesions, fulfilling 2017 Mc Donald's diagnostic criteria for multiple sclerosis. After the careful exclusion of differential diagnoses, she was given pulsed IV methylprednisolone for 5 days, followed by oral teriflunomide. She improved partially, and at 2 months following discharge, she was tolerating the medication well with no further relapses.I. INTRODUCTION Multiple sclerosis (MS) is the commonest chronic, inflammatory demyelinating disease that affects the central nervous system . The worldwide prevalence varies between countries, and it primarily affects people of European descent. It is rare among Asians, black, and Native Americans . In Malaysia, a single-centred study conducted at the largest tertiary hospital in Malaysia found that MS was uncommon, with only 104 patients diagnosed in between 2008 to 2013. They were predominantly females, with a 5:1 ratio, with Malays and indigenous people affected more commonly. Similar to studies on Caucasian, it affects mostly young adults with a mean age onset of 29 years old . Herewith we report a young patient who presented with recurrent vague symptoms over a few months' duration, the diagnostic approach, as well as challenges associated with reaching an early diagnosis.
II. CASE REPORT A 23-year-old female software developer with no known medical illness, first presented to the orthopaedic clinic in June 2018, with right lower limb numbness extending to the right hip, which was exacerbated by walking. Magnetic resonance imaging (MRI) was done to exclude lumbar nerve root compression. Since it was normal and the symptoms improved, she was discharged and reassured without further follow-up. Eight months later, she presented again with bilateral lower limb numbness, which was worse on the right side, extending to bilateral hips. She denied any pain, weakness, or changes in bladder or bowel function. There was no history of fever, cognitive changes, visual disturbances, dysarthria, dysphagia or fatigue. Reviews of other systems were generally normal. There was no family history of neurological or autoimmune disease. She was admitted to the orthopaedic ward and MRI lumbosacral was repeated, showing only minimal posterior disc bulge of the lumbar spine, without significant spinal stenosis or nerve root impingement. She was referred to the neuromedical team for further evaluation. The neuromedical assessment revealed that the patient complained of numbness for the past three weeks. Since then, the numbness has ascended to the bilateral chest. A neurological examination revealed patchy pinprick sensory loss over the left hemithorax up to the neck level, as well as bilateral anterior thighs, inconsistent with any peripheral nerve or dermatomal distribution. Gait, coordination, and the remainder of the motor examinations were all normal. The Romberg sign was not present. Deep tendon reflexes were present and normal, and the plantar response was flexor bilaterally. Otherwise, her vital signs were normal, and the rest of the systemic examination, (cardiovascular, respiratory, and abdominal examination) were also normal. With the new neurological findings, albeit inconclusive, we obtained a repeat MRI to include the cervical and thoracic region, as well as nerve conduction study (NCS). The NCS results revealed L5-S1 radiculopathies. An MRI of the thoracolumbar region showed a short enhancing lesion at T6/T7 with mid cord oedema cranially. The brain MRI showed multiple white matter lesions which are hyperintense on T2W/FLAIR, seen at the right corona radiata, bilateral parietal lobes, and right periventricular region (Figure 1 ). A lumbar puncture and cerebrospinal fluid (CSF) examination were also arranged to exclude other differential diagnoses. A CSF fluid examination revealed clear, acellular fluid with normal protein and sugar. Bacterial latex agglutination was negative and CSF oligoclonal band was not detected. Serum Aquaporin-4 receptor antibody and Myelin oligodendrocyte glycoprotein (MOG) antibody were also sent, and turned out to be negative. . The updated criteria continue to define the needs to fulfil dissemination in time (DIT) and space (DIS) while stressing the importance of excluding mimicking conditions, especially in regions with lower MS disease burden . In this region, special care needs to be taken to exclude Neuromyelitis Optica Spectrum Disorder (NMOSD), which is a more prevalent demyelination disease with serious implication on long term treatment. Paraesthesia is often described as a numbness or tingling sensation of the body, and is a relatively common symptom. Unintentional compression of the nerve by pressure on the arms and legs may cause transient paraesthesia, which is common. At the time of the patient's first presentation, when she presented with only numbness involving lower limb after walking, spinal nerve root compression may have been given serious consideration. However, it was excluded after a normal lumbosacral MRI finding as well as spontaneous resolution of the symptoms. In the second presentation, however, more serious attention was given due to the recurrence and evolution of the symptoms to involve not only the lower limbs, but also the trunk and upper limbs. Without specific signs or symptoms to support a specific diagnosis, the possible diagnoses considered were quite broad. In a young woman, few differential diagnoses were high in the list, such as vasculitis affecting peripheral nerves due to systemic lupus erythematosus, as well as demyelinating diseases, such as multiple sclerosis or NMOSD . Sensory symptoms are the first clinical manifestation in up to 43% of patients with MS, and are primarily caused by myelitis or brainstem syndromes . Other causes that have been considered and excluded include metabolic disorders such as diabetes mellitus, hypothyroidism, uraemia, vitamin B12 deficiency and porphyria. No history suggests the use of implicated agents such as alcohol, drugs, or other toxins. Cancers may also cause nerve compression or may be associated with paraneoplastic syndrome, although it is unlikely in this case. Infectious disease such as Lyme disease is rare in this region, and was much lower in the list, as patients have no exposure or visits to endemic areas. In this case, fortunately, by application of the 2017 McDonald Criteria, an MRI was able to substitute clinical findings in determination of DIS and DIT for diagnosis of MS. This has been extremely helpful, since the patient's presenting complaint provides little clues to enable lesion localization on the central nervous system. The MRI has been used increasingly to aid in the diagnosis of multiple sclerosis, as well as to search for atypical radiological features which are against it . A standardised MRI protocol for brain and spinal cord imaging was proposed for the evaluation of patients with clinically definite or suspected multiple sclerosis to help with the diagnostic process, prognosis and as baseline for follow up scans [6, 7] . In our centre, interferon beta is the only available injectable treatment, and the patient was started on teriflunomide, an oral pyrimidine synthesis inhibitor which has been proven to reduce the annual relapse rate by 34% . Side effects reported by this medication include headache, diarrhoea, hair loss, transaminitis, leukopenia, paraesthesia as well as infections [9, 10] . After 2 months of taking the prescribed medication, the patient did not show any signs of significant adverse effects, which encouraged her compliance to medication. She also reported no further relapse of her symptoms.
IV. CONCLUSIONS Paraesthesia is a potentially alarming symptom that should be taken seriously, and merits further assessment to rule out any possible local as well as systemic diseases such as multiple sclerosis. It is hard to imagine debilitating neurological consequences if we were to continue to dismiss the symptoms or to label these patients as suffering from anxiety or depression. While diagnosis is not always straightforward, the availability of MRI imaging has been tremendously helpful to demonstrate lesions which otherwise cannot be picked up based on clinical presentation alone. CONSENT TO PARTICIPATE Written informed consent was obtained from the patient for the anonymized information to be published in this article. MJoSHT 2022 , 2022 Volume 8, Special Issue on Communicable and Non-Communicable Disease, Multidisciplinary Case Reports. eISSN: 2601-0003 5
Fig. 1 ( 1 Fig. 1(a) shows a hyperintense lesion over the left corona radiata on T2/FLAIR MRI sequence, which shows postcontrast enhancement on the T1 sequence 1(b).
Fig. 2 ( 2 Fig. 2(a) shows a hyperintense lesion over the right corona radiata and periventricular region on T2/FLAIR MRI sequence which did not enhance on T1 sequence 2(b). In this case, dissemination in time (DIT) of MS can be demonstrated by a simultaneous presence of gadolinium-enhancing (image 1b) and non-enhancing lesions (image 2b) simultaneously.
Fig. 3 3 Fig. 3 shows a short segment hyperintense lesion involving the spinal cord. Dissemination in space (DIS) is demonstrated by T2-hyperintense lesions in over two of four typical areas of the central nervous system, (2a): periventricular and juxtacortical lesions, and (3): a spinal cord lesion
|
10.29313/jrpwk.v1i1.75
|
cc-by
| null | null |
openalex
|
This research on pedestrian in the old area of Bandung city of Bandung is aimed at overcoming the problem of the bad pedestrian path in the area of area of Bandung city of Bandung. The existence of the old city area of Bandung which is close to the new market can attract a lot of visitors' interest, unfortunately the condition of the existing pedestrian infrastructure in this area is inadequate. The number of street vendors who sell on pedestrian lanes, motorized parking on the sidewalks, uneven pedestrian lanes, to the lack of pedestrian facilities makes visitors less comfortable to move in this area. The concept of the theory that the authors apply to overcome the problem of pedestrian paths in this region is the concept of livable street. The author expects the theory of urban design and the concept of livable street as being able to overcome the problems regarding the pedestrian path in the old area of Bandung City. The method that I use is a qualitative approach by observing issues that exist and then assessing the criteria for the concept of livable street through the study of literature, then the process of analysis of pedestrian flows, the level of pedestrian services, pedestrian path width requirements, and analysis of variable variable from the concept of liveable street. This research was conducted to solve problems in pedestrian routes that are not liveable in the old Chinatown district of Bandung city.A. Pendahuluan Keberadaan Pasar Baru sebagai pusat perdagangan menjadikan kawasan pasarbaru memiliki peran yang cukup penting bagi Kota Bandung. Kawasan Pasar Baru ini dibangun pada tahun 1896 untuk menampung pedagang yang membuka usahanya di sekitar Alun-alun dan Sumedangweg (Jl. Otista sekarang). Kawasan pasar baru Kota Bandung lebih dikenal sebagai kawasan Pecinaan lama. Kawasan pecinaan lama Kota Bandung ini merupakan sebuah kawasan pusat perdagangan atau komersil yang ramai dikunjungi orang untuk berbelanja segala kebutuhan rumah tangga. Keadaan ini membuat kawasan pecinaan lama dipenuhi pengunjung setiap harinya dari yang datang menggunakan moda transportasi publik maupun menggunakan kendaraan pribadi, pengunjung dari dalam Kota Bandung maupun dari luar kota bahkan dari luar negri, dan banyak mengundang para pedagang kaki lima untuk berjualan di trotoar yang berada di kawasan pecinaan lama Kota Bandung. Jalur pedestrian merupakan ruang publik yang semestinya diciptakan untuk masyarakat dalam melakukan perjalanan dari tempat A ke tempat B dengan berjalan kaki. Akan tetapi jalur pedestrian yang diprioritaskan untuk para pejalan kaki tidak di indahkan oleh beberapa pihak untuk kepentingan pribadi. Kepentingan pribadi yang dimaksud dalam kasus ini berupa pedagang kaki lima yang berjualan atau mendirikan lapaknya diatas jalur pedestrian, ada pula masyarakat yang memarkirkan kendaraan bermotornya diatas jalur pedestrian, dan ada juga para pemilik toko yang masang barang display toko di atas jalur pedestrian. Selain permasalahan diatas, jalur pedestrian dalam wilayah studi ini juga kurang terawat dan seakan diabaikan oleh pemerintah Kota Bandung. Hal ini terlihat dengan banyaknya jalur pedestrian yang rusak dan berlubang, kurangnya penerangan pada malam hari, kurangnya vegetasi yang ditanam di sekitar wilayah studi sehingga kawasan tersebut terasa sangat panas dan gersang pada siang hari, dan minimnya fasilitas sarana jalur pedestrian/ruang pejalan kaki. Dengan adanya permasalahan ini akan menghambat kegiatan pergerakan orang maupun barang yang terjadi di kawasan pecinaan lama Kota Bandung.
B. Landasan Teori Livable street memiliki beberapa prinsip dalam penerapannya, yaitu: menyediakan pilihan transportasi yang banyak, meningkatkan jumlah perumahan yang terjangkau, meningkatkan daya saing ekonomi, adanya dukungan dari masyarakat dalam menciptakan jalan yang livable, mengkoordinasikan dan meningkatkan kebijakan dan investasi, meningkatkan nilai kepedulian masyarakat terhadap lingkungan.
Ruang Jalan Harus Aman dan Nyaman 1. Lalu Lintas Menentukan jenis jalan dengan kapasitas dan karakter jenis jalan untuk dimasukkan dalam skema adalah penting, pendekatan teknik ini didasarkan pada penentuan klasifikasi jalan. Jalan yang aman memiliki 2 fungsi yang aman ( Appleyard, Donald, 1981) yaitu: Mengontrol kebiasaan dari traveller, beberapa karakteristik yang bisa mengontrol volume lalu lintas, komposisi, kecepatan dan kebiasaan adalah tikungan atau jalan yang lurus, blok yang panjang, lereng, lebar, penanda jalan, rambu-rambu, merubah trotoar, tanjakan jalan, zebra cross, garis jalan, landscape, dan beberapa aspek lainnya untuk membuat karakter jalan. Melindungi penduduk dan memberikan kompensasi, perubahan dari emisi lalu lintas (kebisingan, getaran dan polusi), bisa merubah karakteristik dari lingkungan di jalan. Beberapa faktor kritis yang paling mempengaruhi yaitu bertambahnya lereng jalan, bertambahnya kecepatan pada penurunan dari bukit, kebisingan, getaran, polusi udara.
Pejalan Kaki Jalan tempat masyarakat beraktivitas dan tidak ada pengendara yang ugal-ugalan. Anak-anak harus bisa berjalan atau bermain sepeda dengan aman pada jalan lokal di sekolahnya, tempat berhenti bus sekolah, playground, dan parkir untuk para pengunjung. Pengendara mobil, mobil antar-jemput, dan kendaraan lainnya harus mengerti bahwa terdapat teritori di pedestrian. Ketika mengunjungi jalan ini dan oleh karena itu harus dengan pelan dan hati-hati. 1. Jalan yang livable memiliki lingkungan sehat. Donal Appleyard 1981 mengatakan bahwa lingkungan jalan tidak boleh ribut atau ada getaran pada lalu lintas. Penduduk bisa tidur pada waktu siang dan malam dengan tenang. Kemudian mereka bisa menghirup udara segar dan tidak ada debu pada pintu dan jendelanya. Lingkungan jalan harus memiliki tempat dimana orang-orang bersosialisasi.
Ruang jalan sebagai ruang komunitas Jalan harus memiliki tempat yang menjadi ruang komunal, dimana orang-orang bisa merasa senang kemudian mereka bisa bersosialisasi dengan mudah dan tidak terdapatnya orang asing. Jalan umum harus bisa menyaring keanehan yang terjadi pada kehidupan di kota, jalan bisa mendukung aktivitas penduduk, menjaga jalan tetap bersih (Appleyard, D 1981).
Jalan Memiliki Teritori yang Ramah Jalan yang memiliki teritori yang ramah maksudnya adalah dimana penduduk saling melindungi ketika seseorang itu sedang tinggal sendiri, kemudian menjaga privasi orang lain, dimana penduduk itu merasa tidak sendiri dan penduduk itu mempunyai rasa tanggung jawab dan kebanggaan. Jika semua hal ini terjadi maka penduduk itu akan lebih menjaga daerah mereka sendiri, termasuk juga tanamannya, lalu mengawasinya dan penduduk saling menyapa (Appleyard, D. 1981).
Jalan yang unik sebagai jalan yang memiliki nilai sejarah Orang-orang bangga karena memiliki identitas yang spesial pada tempatnya.Identitas yang unik bisa terlihat dari bagian rumah, sungai kecil dan pohon tua (Appleyard, Donald. 1981), oleh karena itu pentingnya place theory dalam spasial design yaitu pemahaman tentang culture dan karakteristik suatu daerah yang ada menjadi ciri khas untuk digunakan sebagai salah satu pertimbangan agar penghuni (masyarakat) tidak merasa asing di dalam lingkungannya.
Ketentuan teknis Jalur pejalan kaki (trotoar) 1. Lebar efektif lajur pejalan kaki berdasarkan kebutuhan satu orang adalah 60 cm dengan lebar ruang gerak tambahan 15 cm untuk bergerak tanpa membawa barang, sehingga kebutuhan total lajur untuk dua orang pejalan kaki bergandengan atau dua orang pejalan kaki berpapasan tanpa terjadi persinggungan sekurang-kurangnya 150 cm. perumahan 1,20 sekolah 1,50 pertokoan dan pusat-pusat 2,00 perbelanjaan terminal dan pemberhentian 1,50 bis/angkot pusat-pusat perkantoran 1,50 pusat-pusat hiburan 2,00 pusat-pusat kegiatan sosial 1,50 daerah industri 2,00 jembatan dan terowongan 1,20 Sumber : Pedoman perencanaan fasilitas pejalan kaki Kementrian Pekerjaan Umum dan Perumahan Rakyat No.02/SE/M/2018
C. Hasil Penelitian dan Pembahasan Berdasarkan perhitungan arus pejalan kaki, maka kita dapat melihat tingkat pelayanan pejalan kaki dengan menggunakan perhitungan berikut.
Banceuy Kondisi jalan banceuy memiliki lebar trotoar 1,5m dengan volume pejalan kaki puncak dalam 15 menit yaitu 100 org/15min pada pukul 12:45 -13:00. Maka data tersebut dapat dimasukan dalam persamaan berikut untuk menentukan tingkat pelayanan pejalan kaki. Vp = V15 15 × We → 100 15 × 1,5 = 4,4 ped/min Berdasarkan hasil perhitungan dan tabel 2.7 tentang tingkat pelayanan pejalan kaki pada ruas jalan Banceuy termasuk kedalam kategori LOS A.
Pecinaan Lama Kondisi jalan Pecinaan lama memiliki lebar trotoar 1,5m dengan volume pejalan kaki puncak dalam 15 menit yaitu 192 org/15min pada pukul 12:15 -12:30. Maka data tersebut dapat dimasukan dalam persamaan berikut untuk menentukan tingkat pelayanan pejalan kaki. Vp = V15 15 × We → 192 15 × 1,5 = 8,53 ped/min Berdasarkan hasil perhitungan dan tabel 2.7 tentang tingkat pelayanan pejalan kaki pada ruas jalan Pecinaan lama termasuk kedalam kategori LOS A.
Gang suniaraja Kondisi jalan Gang suniaraja memiliki lebar trotoar 1m dengan volume pejalan kaki puncak dalam 15 menit yaitu 232 org/15min pada pukul 12:30 -12:45. Maka data tersebut dapat dimasukan dalam persamaan berikut untuk menentukan tingkat pelayanan pejalan kaki. Vp = V15 15 × We → 232 15 × 1 = 15,5 ped/min Berdasarkan hasil perhitungan dan tabel 2.7 tentang tingkat pelayanan pejalan kaki pada ruas jalan Gang suniaraja termasuk kedalam kategori LOS A.
ABC Kondisi jalan ABC memiliki lebar trotoar 1m dengan volume pejalan kaki puncak dalam 15 menit yaitu 144 org/15min pada pukul 12:30 -12:45. Maka data tersebut dapat dimasukan dalam persamaan berikut untuk menentukan tingkat pelayanan pejalan kaki. Vp = V15 15 × We → 144 15 × 1,5 = 6,4 ped/min Berdasarkan hasil perhitungan dan tabel 2.7 tentang tingkat pelayanan pejalan kaki pada ruas jalan ABC termasuk kedalam kategori LOS A.
Alkateri Kondisi jalan Alkateri memiliki lebar trotoar 1m dengan volume pejalan kaki puncak dalam 15 menit yaitu 65 org/15min pada pukul 12:00 -12:15 dan 12:45 -13:00. Maka data tersebut dapat dimasukan dalam persamaan berikut untuk menentukan tingkat pelayanan pejalan kaki. Vp = V15 15 × We → 65 15 × 1,5 = 2,8 ped/min Berdasarkan hasil perhitungan dan tabel 2.7 tentang tingkat pelayanan pejalan kaki pada ruas jalan Alkateri termasuk kedalam kategori LOS A.
Kebutuhan lebar trotoar Berdasarkan hasil analisis tingkat pelayanan LOS pejalan kaki didapati ternyata tingkat pelayanan pejalan kaki di keseluruhan kawasan pecinaan lama Kota Bandung termasuk ke dalam kategori LOS A. Akan tetapi lebar trotoar yang ada di kawasan pecinaan Kota Bandung ini tidak memenuhi standar lebar trotoar yang telah ditetapkan oleh Kementrian Pekerjaan Umum dan Permukiman Rakyat dengan lebar minimal untuk kawasan pertokoan dan pusat perbelanjaan yaitu 2m. Berdasarkan fakta di lapangan tersebut, maka akan dilakukan perhitungan kebutuhan lebar trotoar di kawasan pecinaan Kota Bandung dengan persamaan berikut.
W
Pedagang Kaki lima Keberadaan pedagang kaki lima di kawasan pecinaan lama Kota Bandung ini tersebar hampir ke seluruh kawasan. Berdasarkan data yang penulis peroleh dari survey dapat di ketahui jumlah pedagang kaki lima yang memiliki lapak menetap lebih sedikit dari pedagan kaki lima yang bergerak/dapat berpindah. Untuk mengatasi permasalahan pedagang kaki lima yang berjualan di atas trotoar dan di badan jalan, maka dilakukan relokasi pedagan kaki lima yang tersebar ke sebuah lokasi sehingga para pedagang kaki lima dapat terorganisir degnan baik dan tidak mengganggu pengguna trotoar.
Penerangan jalan Berdasarkan dari identifikasi yang telah dilakukan dapat diketahui bahwa penerangan jalan pada Kawasan Pecinaan Lama tidak semua lampu berfungsi dengan baik, hal ini memberikan kesan yang tidak aman bagi pengguna jalan maupun pengguna jalur pedestrian terutama pada malam hari. Permasalahan ini dapat diselesaikan dengan memberikan perawatan yang baik dan rutin terhadap fasilitas penunjang jalur pejalan kaki. 2. Penghitungan lebar trotoar minimal menggunakan Persamaan : arus pejalan kaki < 16 orang/menit/meter, atau dapat berupa daerah lainnya (Sumber : Pedoman perencanaan fasilitas pejalan kaki Kementrian Pekerjaan Umum dan Perumahan Rakyat No.02/SE/M/2018) Berdasarkan Pedoman perencanaan fasilitas pejalan kaki Kementrian Pekerjaan Umum dan Perumahan Rakyat No.02/SE/M/2018, berikut adalah standar lebar trotoar sesuai dengan penggunaan lahannya. Tabel 2. Standar Lebar Trotoar Lebar minimum mutlak, c (m) Penggunaan Lahan di sekitarnya W = V 35 + N Keterangan: W : adalah lebar efektif minimum trotoar (m) V : adalah volume pejalan kaki rencana/dua arah (orang/meter/menit) N : adalah lebar tambahan sesuai dengan keadaan setempat (meter) ditentukan dalam Tabel 1 Tabel 1. Nilai N N (meter) Keadaan 1.5 Jalan di daerah dengan bangkitan pejalan kaki tinggi * 1.0 Jalan di daerah dengan bangkitan pejalan kaki sedang 0.5 Jalan di daerah dengan bangkitan pejalan kaki renda* Keterangan: * arus pejalan kaki > 33 orang/menit/meter, atau dapat berupa daerah pasar atau terminal arus pejalan kaki 16-33 orang/menit/meter, atau dapat berupa daerah perbelanjaan bukan pasar *
maka di ketahui bahwa tingkat pelayanan pejalan kaki di kawasan pecinaan lama Kota Bandung tergolong pada kategori LOS A yang dimanabesar arus pejalan kaki <16 pedestrian/menit/meter. para pejalan kaki dapat berjalan dengan kecepatan yang relatif cepat. Berjalan cepat yang dimaksud adalah berjalan pada bahu jalan karena trotoar yang seharusnya digunakan oleh pejalan kaki telah dimanfaatkan oleh pedagang kaki lima dan parkir kendaraan bermotor. Meskipun di semua ruas jalan yang ada di kawasan lama Kota Bandung termasuk kedalam Kriteria tingkat pelayanan pejalan kaki LOS A, akan tetapi lebar trotoar tidak sesuai dengan spesifikasi yang telah dibuat oleh Kementrian Pekerjaan Umum dan Perumahan Rakyat. Dari hasil perhitungan yang telah dilakukan dapat diketahui bahwa pelebaran lebar trotoar dikawasan pecinaan lama Kota Bandung masih kurang dari standar yang telah ditetapkan oleh Kementrian Pekerjaan Umum dan Perumahan Rakyat, yang dimana kawasan pecinaan lama Kota Bandung merupakan sebuah kawasan perdagangan/perbelanjaan yang harus memiliki lebar trotoar minimal 2m, Namun pada fakta di lapangan lebar maksimal trotoar di kawasan pecinaan lama Kota Bandung hanya 1,5m. Maka dari itu penulis menetapkan bahwa lebar trotoar yang ada di kawasan pecinaan lama Kota Bandung harus diperlebar sebesar 50cm sehingga memenuhi standar yang ada. 4. Jalan ABC W = V 35 + N → 15 35 + 1 = 1.42m 5. Jalan Alkateri W = V 35 + N → 2 35 + 1 = 1.05m D. Kesimpulan Ruang jalan yang aman dan nyaman 1. Kebutuhan Lebar Trotoar Setelah dilakukan penelitian = V 35 + N Keterangan : W : Lebar efektif trotoar V : Volume Maksimum pejalan kaki N : 1 (Nilai koefisien pada Tabel 1, meskipun nilai 1 pada tabel tidak sesuai dengan persyaratan nilai arus pejalan kaki, tetapi kawasan pecinaan lama Kota bandung termasuk kedalam kawasan perbelanjaan sehingga tetap mendapat nilai koefisien 1). 1. Jalan Banceuy W = V 35 + N → 7 35 + 1 = 1.2m 2. Jalan Pecinaan Lama W = V 35 + N → 12 35 + 1 = 1.34m 3. Jalan Gang Suniaraja W = V 35 + N → 8
|
10.54097/fbem.v7i1.3961
|
cc-by
| null | null |
openalex
|
The development of the beef cattle business in Jiaxian County, Henan Province is currently facing issues such as a lack of funding, a lack of technology, a lack of brands, and a low relative efficiency of the industry, according to this study, which provides a detailed introduction to the situation. This has significantly impeded the county-level beef cattle industry's breakthrough development in Henan Province. It is proposed that measures such as increasing government funding for key links, innovating financial and insurance measures, improving technical service system construction, vigorously supporting animal husbandry technology innovation, incubating and cultivating local well-known brands, creating and improving the entire industrial chain and innovative interest linkage mechanism, and so on be implemented. To serve as a model for accelerating the breakthrough development of the beef cattle industry in Jia County, Henan Province.Introduction With the continuous improvement of China's national economic level, the consumption capacity and consumption level of the people have also been further improved. Beef, as the third most consumed meat product in the world, has gradually been recognized by more and more Chinese people. Since 2000, the proportion of beef in my country's meat consumption has been continuously increasing, and the quality, grade and food safety of China's beef cattle industry are also facing new challenges. Jiaxian County, Henan Province is a large grain county and is famous for its Jiaxian Red Cattle, one of the eight fine-bred yellow cattle in China. The beef cattle breeding industry is also an important part of increasing farmers' income in Jiaxian County (Lei, 2019) . Therefore, it has profound practical significance to ensure the high-quality development of the beef cattle industry in Jiaxian County.
Practical Significance of Developing Beef Cattle Industry in Jia County
Promoting the high-quality development of the red cattle industry in Jiaxian County Jia County has placed a high value on the development of the Red Bull industry in recent years, and has issued a series of relevant policy documents that laid a policy foundation for the Red Bull industry in Jia County, as well as a policy basis for the high-quality development of the Red Bull industry in Jia County. Jiaxian County red cattle industry research can effectively promote high-quality development of the red cattle industry in Jiaxian County. (Bell & Harley, 2022) .
Improve the Industrialization Management Level of Red Bull Industry in Jiaxian County The improvement of the red cattle industry's industrial structure, extension of the industrial chain, promotion of the integration of the local agricultural primary, secondary, and tertiary industries, as well as an increase in the level of industrialised management, can all be achieved by fostering the development of the red cattle industry in Jiaxian County. (Ivanov & Sokolov, 2019)
Promoting income increase of farmers in
Jia County Jia County, a typical large traditional agricultural county in central Henan, with a sizable rural poor population, urbanrural inequalities, and a persistent wealth disparity, all of which contribute to social instability. In addition to raising farmers' income and enhancing their quality of life and living standards, promoting the development of the red cattle sector in Jiaxian County has the potential to reduce the gap between social groups in terms of income distribution and access to public amenities. Enhance the infrastructure of the red cattle breeding communities in Jiaxian County, transform cow manure into field treasures, and advance the development of a peaceful society there. (Halala, 2020) .
Provide a basis for scientific decisionmaking by government departments At the moment, the development of the red cattle industry in Jiaxian County is limited. Through research on the development of the red cattle industry in Jiaxian County, this paper identifies problems and causes in the development process and proposes appropriate countermeasures. It is beneficial to the development of the red cattle industry in Jiaxian County, and it can also serve as a theoretical foundation for the Jiaxian government's and relevant departments' scientific decision-making. In short, the development of Jiaxian's characteristic red cattle industry is an important driving force to improve the income and living standards of farmers in Jiaxian(Jacob et al., 2021).
Problems Existing in the Development of Beef Cattle Industry in Jia County
Lack of funds, not many channels for industrial financing On the one hand, the initial investment required in the beef cattle industry is substantial. The initial one-time investment is at least 100,000 yuan, which includes the cost of calves as well as comprehensive expenditures such as pens, manure treatment facilities, and epidemic prevention in the early stages. Furthermore, the supply of seed sources in Jia County is insufficient, and the red cattle varieties are mixed, raising the cost of breeding inputs even further. Farmers in Jiaxian County are under a lot of investment pressure, and they face a lot of uncertainty due to epidemic diseases, which makes most farmers discouraged. On the other hand, the loan requirement is rather large, bank loans demand and freshly constructed pens and transferred land typically cannot be utilised as mortgage assets, making it challenging for owners to get loans. Credit loans often have minimal amounts and a period of two years or less. However, the beef cattle industry has a lengthy investment return period, making short-term profit returns challenging. Additionally, Jiaxian County's insurance programme for the beef cattle business is not perfect, and there are not many claims. Typically, a deceased cow's compensation is only a few thousand yuan, which is far from sufficient to make up for the loss, and the risk of breeding is uncontrollable (Pohlmann et al., 2020) .
Lack of technology, technical service system is not perfect On the one hand, the majority of the Jia County beef cow breeding staff are descendants of farmers who abandoned the countryside. The labour force is often elderly, and knowledge is slowly updating. It is challenging to spread the use of sophisticated breeding management approaches. Jiaxian County, on the other hand, is devoid of academic research centres. The Jiaxian Red Cattle Breeding Demonstration Experimental Base only listed the Jiaxian Red Bull Research Institute in July 2022. This organisation is exposing talents and developing collaboration platforms because it is still in its infancy.It is difficult to achieve technological innovation in the short term, and the role of personnel training, technical services, and demonstration drives is also very limited. In addition, after the adjustment and reform of township divisions, animal husbandry and veterinary stations have not been retained, and grassroots animal husbandry and veterinary professional technicians are scarce, and some of them are part-time jobs. Even more weak technical services.
Lack of brand, low level of industry promotion A good brand effect can provide significant economic and social benefits to a company, and it is the number one priority in the development and promotion of an industry. Jiaxian Red Bull's brand building is currently low, the brand effect has not been realised, and it lacks a competitive advantage in the face of homogeneous competition. One the one hand, Jiaxian Red Bull's brand recognition isn't very high. Although Jiaxian Red Bull is a well-known brand of beef cow to the majority of residents in Jiaxian County and the neighbourhood. Jiaxian Red Bull does not have a competitive edge over other homogeneous beef cow brands in provinces with larger territories, developed regions, or even China because of its low brand awareness in these areas.On the other hand, Jiaxian Red Bull's brand-building strategy remains unclear. Despite having goods and names with distinctive advantages, like Jiaxian Red Bull. However, neither the government nor businesses currently have any well-defined plans on how to increase brand recognition and scale up Jiaxian Red Bull product promotion. The Jiaxian Red Bull brand has to be developed, but there is an urgent need to address the lack of appropriate capital investment and methodical development strategy. (Lei, 2019).
Relatively low efficiency, weak protection of industrial factors On the one hand, the beef cattle industry has gradually lost its economic advantages in all facets of the business. The red cow breed is experiencing a provenance issue as a result of the modest size of red cattle breeding in Jiaxian County and the inconsistent supply of lineage. Additionally, there is a widespread lack of pasture of excellent quality, and feeding red cattle has a high breeding cost. No regional superior brand has emerged during the sales process as a result of the absence of a production and sales platform.Most of the beef cattle in Jia County mainly rely on dealers or middlemen for internal purchase and export, and the profit margin is further greatly reduced.In the processing link, in 2022, there will be only one large-scale slaughtering and processing plant for beef cattle in Jiaxian County, with an annual slaughtering and processing capacity of only tens of thousands of head, a serious shortage of slaughtering and processing capacity, a smaller proportion of intensive processing, a short industrial chain, weak antirisk ability, and low added value . Jia County's beef cattle are primarily bought and sold internally by traders or middlemen, which further reduces the profit margin. In Jiaxian County, there will only be one large-scale plant for slaughtering and processing beef cattle by 2022, with an annual capacity of only tens of thousands of head. This will result in a severe shortage of capacity for slaughtering and processing, a smaller proportion of intensive processing, a short industrial chain, weak anti-risk ability, and low added value. (Leuschner & Charvet, 2013) .
Countermeasures to Promote Highquality Development of Beef Cattle Industry in Jiaxian County
Increase government support and strengthen industrial policy guarantees The first is to strengthen industry financial support. The county government should increase subsidies, formulate preferential policies, and guide social capital to increase investment in all links of the industrial chain. The county government should integrate project funds, form a joint force, establish a diversified investment mechanism, improve the construction of investment and financing platforms, solve funding problems, and help the development of the beef cattle industry. The second is to innovate financial insurance measures. Guide financial institutions to innovate and develop special financial service products for the beef cattle industry, relax mortgage and pledge guarantee conditions, revitalize the assets of the beef cattle industry, moderately increase the amount of compensation, and help breeding owners reduce risks. And through government financial subsidies for premium funds, insurance companies to underwrite claims, and encourage farmers to actively participate in insurance, build and improve risk protection, and gradually improve the insurance mechanism for beef cattle breeding. The third is to guarantee land for the development of beef cattle industry. The land used for the construction of breeding production and its directly related agricultural facilities.
Strengthen the innovation of animal husbandry technology and improve the technical service system One is to vigorously encourage technological innovation. Formulate incentive measures, give moderate preference to the approval and recommendation of scientific research projects, encourage scientific research institutions and enterprises to carry out scientific and technological research in key links of the industrial chain, and improve scientific and technological content to promote industrial upgrading. The second is to make the scientific research team bigger and stronger. Jiaxian agricultural scientific research institutions should continue to introduce professional scientific research talents in the field of beef cattle, strengthen the scientific research team, and improve scientific research capabilities. Deepen cooperation with institutions of higher learning and scientific research institutions. Focus on key links such as multiple beef cattle breeding, breed selection, and disease prevention and control to refine cooperation projects. Realization cooperation measures to jointly tackle key problems, and establish a long-term and stable scientific research cooperation platform. Improve scientific research innovation and achievement transformation capabilities. The third is to improve the technology service system. It is necessary to strengthen the introduction and cultivation of grassroots professional talents. And encourage enterprises and cooperatives to carry out paid technical services and grassroots talent training, and establish and improve the animal husbandry technology service system (Liu & McKinnon, 2016) .
Cultivate local well-known brands and enhance regional competitiveness of industries "Brand is an inevitable choice for market development" (Qu, 2017). Brand is the long-term driving force of industrial development and a symbol of improving market competitiveness. It is imperative for Jia County to make a breakthrough in the development of the beef cattle industry and build a well-known brand in the beef cattle industry. It is imperative to enhance the added value of products and the competitiveness of the industrial region. On the one hand, beef cattle breeding should be further regulated. Increase human and material support for the introduction and promotion department, cultivate and expand local beef cattle breeding leading enterprises, improve the level of breeding technology, expand the scale of breeding, and ensure the supply of provenance. It is necessary to unify the technical standards and meat quality standards of the main varieties, and establish a unified and good industrial brand image. On the other hand, we must vigorously promote and cultivate high-quality brands. Digging deeply into Jiaxian Red Bull culture, packaging and planning brand trademarks. Comprehensively promote green and healthy breeding technology, strengthen the quality supervision of beef cattle products, expand organic product certification, actively cultivate corporate product brands and regional public brands, vigorously carry out brand marketing recommendations, and continuously improve the market influence of beef cattle products (Wang & Chen, 2017).
Create and improve the entire industrial chain, and innovate the mechanism of linking interests One is to keep increasing the size of farms. Accelerating the promotion and implementation of cutting-edge feeding and management methods for beef cattle is important in addition to increasing the scale and breeding capacity of local beef cattle. Increase market share, strengthen the front end of the industrial chain, aid in the expansion and quickening of the beef cattle industry, and provide resource assurance for the construction of the complete industrial chain.The second is to significantly increase the capacity of beef cattle for processing and slaughter. Increased slaughter and processing capacity may not only address the issue of farm product sales, but also strengthen the industrial chain, significantly raise the added value of beef cattle products, increase the industry's resilience to risks, and improve market competitiveness.The third is to create and enhance the system for tying interests together. The government ought to direct businesses to develop a system that closely links the interests of agricultural businesses with those of the upstream and downstream industries. develop a mutually beneficial community of interests made up of "slaughtering and processing firms + breeding enterprises/professional cooperatives + farmers" gradually. Increase the sector's capacity to withstand market threats. Radiation encourages more farmers to join the industrial chain and increases farm income. To attain wealth for everybody, hasten the high-quality growth of the beef cattle sector in Jiaxian County.
|
10.37636/recit.v611427
|
cc-by
| null | null |
openalex
|
In this work, a study dealing with the correlation between the fatigue crack growth behavior and the temperature evolution at the tip of the fatigue crack of the AISI 1018 steel is presented. Both the crack length and the temperature at the crack tip were experimentally obtained from a fatigue test carried out according to the ASTM E647; the temperature at the crack tip was simultaneously acquired by thermocouples and infrared thermography, while the crack length was acquired through a microscope. The experimental data were processed aimed at plotting the curves of the temperature against cycles as well as the crack length against cycles in order to be able to correlate the information and, therefore, to obtain by linear regression of the experimental data a model to correlate the temperature at the crack tip to the crack length. The results showed that, on one hand, the linear regression is in good agreement with the experimental data, and, on the other hand, the proposed model allows estimating the trend and the magnitude of the temperature when the crack grows.Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245
Introducción Los primeros estudios sobre fatiga fueron documentados por Albert desde 1937, y desde entonces hasta la actualidad es un tema de gran interés entre la comunidad científica. Todo este interés tiene su motivación en el hecho de que el fenómeno de fatiga es común en las fallas de materiales. Zerbst et al reportaron que entre el 80 y el 90% de los casos registrados de fallas estructurales tienen a la fatiga como la causa. Esto se manifiesta en estructuras y componentes de máquinas, usadas en áreas como las ingenierías aeroespacial, civil y mecánica . La mayoría de los componentes mecánicos que se someten a cargas cíclicas son susceptibles a desarrollar grietas y eventualmente causar fallas por fractura, que a menudo ocurren sin previo aviso y con consecuencias catastróficas . En la fatiga se desarrollan esfuerzos cíclicos a través del tiempo, que degradan el material hasta el punto en que las pequeñas grietas se nuclean. Después la grieta comienza a propagarse y termina en la ruptura final de la pieza . El estudio de la propagación de grietas por fatiga es importante, debido que de ahí depende la seguridad de las partes mecánicas y estructurales, para evitar accidentes que conducen a pérdidas económicas, humanas y ambientales.
Antecedentes Paris y Erdogan fueron los primeros investigadores que utilizaron el factor de intensidad de esfuerzos para determinar una relación entre la condición de carga, la longitud de grieta y la velocidad de propagación. La ley de París es el tratamiento de resultados de muchos datos experimentales en el marco de la solución elástica del problema de propagación de grietas. Como consecuencia, esta ley no explica la naturaleza física del proceso de propagación de grietas en los metales e inicia muchas discusiones científicas e intentos de derivar una descripción alternativa de la ley de propagación de grietas. Recientemente, se han propuesto enfoques experimentales para evaluar la propagación de grietas [6, 7] , principalmente a partir del desarrollo de las técnicas modernas de medición de la temperatura, la cual, como se reporta ampliamente en la literatura , se puede correlacionar con la propagación de grieta por fatiga debido a que una gran variedad de estudios demuestran que, en la mayoría de los materiales metálicos, la energía de histéresis por deformación plástica se convierte en calor, como se reportó en el trabajo de Meneghetti et al . Por lo anterior, se considera que la mayor parte de la energía mecánica de entrada se disipa en forma de calor y eleva la temperatura del material y, en el caso particular de la etapa de propagación de grieta, existe una distribución de temperatura que alcanza su valor máximo en la vecindad de la punta de la grieta, como lo demostraron Pandey y Chand . Actualmente es posible encontrar trabajos como el de Meneghetti y Ricotta , quienes, a través de la medición de la temperatura en probetas sometidas a fatiga, demostraron que existe disipación de calor en la punta de la grieta, por lo que la medición de temperatura por termografía se ha aplicado ampliamente en la investigación del comportamiento a fatiga en los últimos años . Resultados de trabajos recientes demuestran que, a medida que crece la grieta, hay un aumento de la temperatura superficial de la probeta, lo cual plantea la necesidad de comprender los fenómenos físicos en la propagación de grietas por fatiga desde el punto de vista de la Termodinámica. Un ejemplo es el trabajo de Idris et al , quienes midieron simultáneamente la temperatura y la longitud de la grieta durante los Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245 ensayos de crecimiento de grietas por fatiga hasta la ruptura, bajo condiciones de carga de amplitud constante y variable. Hajshirmohammadi y Khonsari reportaron un modelo matemático para determinar la tasa de propagación de grietas, el cual considera la temperatura superficial de la probeta como variable. Por lo anterior, en el presente estudio se propone la medición de la temperatura en la vecindad de la punta de la grieta durante la propagación de la grieta por fatiga bajo una carga de modo I de amplitud constante para un acero AISI 1018 bajo condiciones de esfuerzo plano para correlacionar el comportamiento del crecimiento de grieta y la evolución de la temperatura en la punta de la grieta.
Metodología Se realizaron 2 ensayos experimentales de crecimiento de grieta por fatiga bajo la norma ASTM E647 , en la que menciona que se debe realizar los ensayos en dos etapas: preagrietamiento y propagación de grietas. En la etapa de preagrietamiento, la norma ASTM E647 especifica que la longitud inicial de pregrieta corresponde a la longitud del centro del orificio de carga hasta la punta de la muesca (10 mm) que se muestra en la Figura 1. En la etapa de propagación de grieta, la longitud de grieta inicial fue de 11.3 mm, que corresponde a la suma de la longitud inicial de pregrieta y el tamaño de la pregrieta. A partir de 11.3 mm se midieron simultáneamente la longitud de grieta y la temperatura en la punta de la grieta en una probeta de tensión compacta (CT, por sus siglas en inglés). Se realizaron intervalos de medición de la longitud de grieta (∆a) de 0.033 mm, debido a que la norma ASTM E647 recomienda que para probetas CT de 40 mm de anchura (w), los intervalos de medición deben ser menor o igual a 1.6 mm. La temperatura se registró en cada intervalo de longitud de grieta con su respectivo número de ciclos.
Materiales y equipo El material utilizado en este estudio es un acero AISI 1018. Este tipo de acero es muy usado en construcción de puentes, tuberías y edificios . El acero es de tipo estructural y de bajo contenido de carbono. La composición química del material se encuentra en la Tabla 1. Las propiedades mecánicas y cíclicas de dicho acero se presentan en la Tabla 2. Tabla 1. Composición química del acero AISI 1018 . Las muestras de tensión compacta (CT) se obtuvieron de una placa de acero AISI 1018 de medidas 10 x 8 x 3/8 de pulgadas, la cual se maquinó para dimensionarla de acuerdo a la norma ASTM E647 para obtener las dimensiones que se muestran en la Figura 1. Se seleccionó la muesca en V y se maquinó mediante el proceso de fresado.
% C % Mn
Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245 Figura 1. Dimensiones de probeta de tensión compacta (CT) en mm. Con el fin de realizar una observación adecuada del crecimiento de la grieta en las probetas, una de las caras de las probetas se pulió en acabado espejo. En la otra cara se realizó un pulido menos fino, con la finalidad de que la rugosidad de la superficie no afectara la trayectoria de la grieta, durante la propagación de la misma. El desbaste se realizó con papeles abrasivos de carburo de silicio, de tamaño de grano grueso a un tamaño de grano muy fino (de lija #100 hasta lija #2000). Se utilizó agua como medio de lubricación y el pulido se realizó de forma manual. La cara menos pulida se recubrió con una fina capa de pintura negra rociada en la superficie para aumentar la emisividad térmica al valor conocido de 0.98. Para garantizar una punta de grieta aguda, fue necesario generar un preagrietamiento sobre la entalla. La pregrieta se generó mediante carga cíclica de tensión-tensión. El manual ASM menciona que para ensayos de tensión-tensión, la relación de carga debe ser mayor a 0, por lo que recomienda una relación de 0.1. También hace mención que las frecuencias de forma de onda sinusoidal son más fáciles de controlar en máquinas de ensayo equipadas con sistemas servo hidráulicos. Las cargas aplicadas se propusieron en ciclos de carga sinusoidal con una frecuencia de 10 Hz y una relación de carga R = 0.1. Para el proceso de preagrietamiento, cada una de las probetas fue sometida a cargas cíclicas en la máquina universal MTS Landmark servo hidráulica de circuito cerrado (ver Figura 2a), con una celda de carga de 100 kN de capacidad (ver Figura 2b), en modo de control de carga en condiciones de amplitud de carga constante, ΔP. La sujeción de las probetas (CT) se realizó en mordazas tipo "clevis", ajustando la altura de las mordazas con el controlador Flextest 40 de la máquina servo hidráulica. Los datos para preagrietamiento de cargas máxima, mínima y promedio, se introdujeron en una computadora mediante el software MTS TestSuite. Con un microscopio óptico Struers modelo PSM-10, se ajustó el punto de observación en la punta de la muesca. La medición de la pregrieta se monitoreó con la regla graduada incorporada en el ocular de medición hasta que la pregrieta alcanzó 1.33 mm de longitud. La longitud final de la pregrieta fue determinada de acuerdo a las indicaciones que especifica la norma ASTM E647 en el numeral 8.3.2, en el cual se sugiere que el valor mínimo que debe tener la pre-grieta, es de 1 mm. Los datos de ensayo para la etapa de preagrietamiento, se muestran en la Tabla 3. Una vez que se finalizó el proceso de preagrietado, se realizaron los ensayos de propagación de las grietas. Se consideró usar la misma frecuencia de 10 Hz y una relación de carga R=0.1, debido a recomendaciones de la norma ASTM E647 . El microscopio óptico se ajustó en la punta de la pregrieta generada, la cual fue la referencia de las mediciones para la etapa de propagación de grieta. Se registraron el número de ciclos cada 0.03 mm de avance de grieta. La prueba se consideró finalizada cuando la probeta se fracturó totalmente. Los datos de cargas máxima, mínima y promedio introducidos en la máquina de ensayos para la etapa de propagación de grieta, se muestran en la Tabla 4.
Medición de crecimiento de grieta La medición de la grieta se realizó de manera visual (ver Figura 2). Se colocó el microscopio al frente de la muestra y con la lente de objetivo (ver Figura 2g) se realizaron observaciones con Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245 aumentos de 50x. El microscopio se desplazaba horizontalmente mediante un carro (ver Figura 2d) para seguir la trayectoria de la punta de grieta. A medida que se registraba cada avance de longitud de la grieta, también se consideraba el registro del número de ciclos.
Medición de temperatura de la grieta La temperatura superficial de la muestra se midió a través de una cámara infrarroja marca Hti modelo HT-18 con resolución de 220 x 160 píxeles y sensibilidad térmica de 0.07 C. La cámara infrarroja se instaló en la parte posterior de la probeta apoyada en un tripié (ver Figura 3d). El proceso de registro de temperatura se hizo en simultaneidad con el crecimiento de grieta mediante captura de imágenes. En otra medición de temperatura, se colocaron termopares tipo K modelo Fluke 52 II (ver Figura 2i). Las terminales de los termopares se fijaron con cinta adhesiva laminar a 1.33 milímetros de la punta de la grieta y el valor de la temperatura se registraba en un monitor. El proceso de registro de temperatura se hizo en simultaneidad con el crecimiento de grieta.
Resultados Después de concluir la prueba de propagación de grietas por fatiga, se notó una clara diferencia en la apariencia física en ciertas áreas superficiales de sección fracturada de la probeta (ver Figura 4). Se encontró que el área fracturada con condiciones de superficie lisa y plana (región a), mostró un crecimiento gradual de la grieta, que es la etapa de propagación de grieta por fatiga. En esta superficie fracturada, no se muestra ninguna marca de playa a simple vista en la región de crecimiento de grieta. Esto demostró, de acuerdo con la literatura , que las marcas de playa en la superficie de la fractura son invisibles cuando se usa la carga de amplitud constante, en comparación con cargas de amplitud variable, que son vistas a simple vista. El área fracturada con condiciones de superficie rugosa e irregular (región b), mostró un crecimiento de grieta inestable y puede caracterizarse como un crecimiento rápido de la longitud de la grieta. Cuando la pieza tiene una fractura repentina y completa (región c), se observó un desprendimiento irregular de material en los extremos del espesor de la probeta y la superficie mostró porosidad. La transición de crecimiento de grieta estable a inestable se puede distinguir notoriamente debido al tipo de superficie liso y rugoso.
Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245 Figura 4. Aspecto de la superficie de fractura de las probetas CT. a) Región de propagación de grieta, b) Región de crecimiento inestable de grieta, c) Fractura repentina de la pieza. En la Figura 5 se muestra la curva de longitud de grieta contra ciclos de fatiga (a-N) para el acero AISI 1018. Los datos experimentales de propagación de grietas por fatiga (ver Figura 5), muestran que la grieta inicialmente creció a un ritmo lento como se reporta en la literatura , y comenzó a acelerarse el crecimiento de grieta después de 150000 ciclos a partir de una longitud de 19 mm aproximadamente. La tasa de crecimiento de la grieta aumentó junto con la longitud de la grieta. Los ciclos totales hasta la falla fueron en promedio de 174773 ciclos bajo las condiciones de carga mostrados en la Tabla 4. El punto final de la curva indica la fractura final de la probeta durante la prueba. Figura 5. Datos experimentales de longitud de grieta vs número de ciclos. La curva de la temperatura respecto al número de ciclos mostrada en la Figura 6 se obtuvo a partir de los datos de termopares, y la cámara termográfica se usó para corroborar datos. La temperatura mostró un incremento lento y constante desde el inicio hasta los 150000 ciclos donde la probeta pasa la mayor parte de su vida de fatiga. Después de los 150000 ciclos, la temperatura aumentó continuamente hasta llegar a la fractura total de la muestra que corresponde 174773 ciclos. Este comportamiento en el incremento estable de temperatura es debido a un equilibrio entre la disipación de calor y la generación de energía por deformación plástica, que ha sido documentado por Meneghetti y Ricotta . El incremento continuo de temperatura representa entre 5 al 10% de la vida de fatiga , ya que un aumento repentino de temperatura representa una pequeña cantidad de ciclos después de los 150000 ciclos. La temperatura registrada en la probeta fue en un inicio con una temperatura de 24.6 C y finalizó con 29.2 C. La diferencia entre las temperaturas inicial y final fue de 4.6 C. Figura 6. Comportamiento de temperatura superficial de la probeta.
Discusión de resultados En este estudio se encontró que la temperatura aumenta a medida que la grieta crece hasta la fractura final de la prueba (ver Figura 7). Esta información se puede utilizar para predecir el punto de transición entre el crecimiento estable e inestable de la longitud de grieta, y que se manifiesta en los incrementos de temperatura de la probeta. En la Figura 8 se muestra la relación del crecimiento de grieta con la temperatura de la superficie de la probeta, la cual revela que existe una relación exponencial desde la etapa de propagación de grieta hasta la fractura. Se observa una curva más pronunciada después de los 18 mm de longitud de grieta debido a la inestabilidad en su crecimiento. El modelo que se muestra en la ecuación (1), donde T representa la temperatura superficial de la probeta, y la longitud de grieta se expresa como a. El modelo se obtuvo mediante la técnica de regresión exponencial. Se determinó el coeficiente de correlación R 2 con un valor de 0.987 para establecer la correlación entre los resultados obtenidos en el presente modelo y los resultados del experimento. T = 29.28 -56.60e -0.21a (1) Figura 8. Comportamiento de la temperatura y la longitud de grieta durante el ensayo experimental. La velocidad de propagación de grieta ( da dt ) mostrado en la Figura 9, presenta una velocidad constante de crecimiento en la etapa de propagación de grieta antes de los 150 000 ciclos. Este comportamiento se presenta como una línea recta (ver Figura 9), donde la velocidad se mantiene constante a 6.78E-4 mm/s. Después de los 150 000 ciclos la velocidad de propagación de grieta se comporta de manera exponencial, debido a que su velocidad aumenta mientras transcurren a pequeños intervalos de ciclos hasta llegar a la ruptura total de la probeta. Paris y Erdogan reportaron que existe un crecimiento acelerado de la tasa de la propagación de la grieta antes de la fractura total de una pieza sometida a cargas por fatiga, por lo que la Figura 9 representa el mismo comportamiento al final de la fractura de la probeta. El ajuste de la curva se obtuvo mediante la técnica de regresión exponencial y se obtuvo el valor de 0.997 para el coeficiente de correlación R 2 . De acuerdo con Sivák y Ostertagová , un valor de R 2 superior a 0.90 se considera muy bueno para evaluar la calidad de regresión. La tasa de temperatura ( dT dt ) mostrado en la Figura 10 tiene la misma tendencia de evolución que la velocidad de propagación de grieta, con un rápido incremento en la etapa final, pero con una gran dispersión de datos en el estado estacionario. Se estableció una tasa de temperatura constante de 0.0002 C /s. Después de los 140 000 ciclos la tasa de temperatura se comporta de manera exponencial, debido a que la velocidad de propagación de grieta aumenta. Hajshirmohammadi y Khonsari reportaron un comportamiento similar de la tasa de temperatura para un acero inoxidable 304. El ajuste de la curva se obtuvo mediante la técnica de regresión exponencial con un coeficiente de correlación R 2 = 0.774, que demuestra poca precisión debido a la dispersión de temperatura mencionada. Figura 10. Comportamiento de la tasa de temperatura. entropía, debido a que la fatiga es un proceso irreversible . La temperatura y la entropía son variables que, al estar relacionados con la longitud de grieta, han sido incorporados en modelos de propagación de grietas o en predicciones de vida a la fatiga reportado en la literatura .
Conclusiones Se encontró un modelo que relaciona el comportamiento del crecimiento de grieta y la temperatura en la punta de la grieta, en la propagación de grietas por fatiga. Las pruebas de propagación de grietas por fatiga, realizadas en el acero de bajo carbono AISI 1018 con una relación de carga (R) de 0.1, muestran un aumento en la temperatura superficial de la probeta a medida que crece la grieta por fatiga cada 0.3 mm. El modelo predice la evolución completa de la temperatura durante el proceso de fatiga, incluido el aumento de la temperatura antes de la falla por fatiga. La comparación de las predicciones del modelo con los resultados experimentales muestra una buena concordancia en la magnitud de la temperatura. Los resultados demuestran que la temperatura es una variable que se puede considerar en modelos de propagación de grietas, debido a su linealidad con respecto al incremento de longitud de grieta que existe exclusivamente en la etapa de crecimiento estable de la grieta. Se utilizaron técnicas de medición de termopares y termografía para conocer mejor la naturaleza de la propagación de grietas, que demostró un ascenso de temperatura en cada intervalo de número de ciclos. Sin embargo, la dispersión de datos en la tasa de temperatura se debe a que se requiere de una mayor precisión en el equipo de medición de temperatura, por lo que se comenta que es un factor importante a considerar en este tipo de estudios. Se presentó un aumento repentino de temperatura al final de la vida de fatiga, acompañado de la fractura total de la pieza. Este comportamiento se puede utilizar como una advertencia inmediata de una falla inminente, y puede ser útil, por ejemplo, para interrumpir el funcionamiento de la maquinaria o reemplazar piezas agrietadas en un proceso de mantenimiento, a fin de evitar fallas catastróficas.
Agradecimientos Al CONACYT por la beca número 1080300. Al Centro de Investigación e Innovación Tecnológica (CIITEC) por el apoyo en las pruebas experimentales en sus instalaciones.
Reconocimiento de autoria Darío Antonio García Lavariega: Borrador original; Escritura; Análisis formal; metodología. Arturo Abúndez Pliego: Revisión y edición, supervisión. Christian Jesús García López: Ideas; supervisión. Jan Mayén Chaires: Ideas. Figura 2 . 2 Figura 2. Configuración con termopares. a) Máquina universal MTS, b) Celda de carga, c) Mordaza, d) Carro, e) Microscopio, f) Ocular de medición, g) Lente de objetivo, h) Botón de lámpara LED, i) Termopar, j) Monitor del termopar.
Figura 3 . 3 Figura 3. Configuración con cámara termográfica. a) Microscopio, b) Probeta, c) Cámara termográfica, d) Tripié.
Figura 7 . 7 Figura 7. Comportamiento de la temperatura y la longitud de grieta durante el ensayo experimental. a) Región estable, b) Región inestable.
Figura 9 . 9 Figura 9. Comportamiento de la velocidad de propagación de grietas.
) 7850 Esfuerzo de fluencia (MPa) 386 Esfuerzo último (MPa) 634 % P % S 0.15 -0.20 0.60 -0.90 0.040 0.05 Módulo de elasticidad (GPa) 200 Relación de Poisson 0.29 Densidad (kg/m 3 Tabla 2. Propiedades del acero AISI 1018 .
Tabla 3 . 3 Condiciones de carga de preagrietamiento. Dato Símbolo Valor Carga máxima, kN Pmáx 6.56 Carga mínima, kN Pmin 0.656 Carga promedio, kN Pprom 3.609
Tabla 4 . 4 Condiciones de carga de propagación de grieta. Dato Símbolo Valor Carga máxima, kN Pmáx 7.01 Carga mínima, kN Pmin 0.701 Carga promedio, kN Pprom 3.855
Revista de Ciencias Tecnológicas (RECIT). Volumen 6 (1): e245 0.014 Datos experimentales Curva exponencial 0.012 (mm/s) 0.008 0.010 da/dt 0.006 0.004 0.002 0.000 0 50000 100000 150000 200000 Número de ciclos 0.0014 Datos experimentales Curva exponencial 0.0012 (c/s) 0.0008 0.0010 dT/dt 0.0006 0.0004 0.0002 0.0000 0 50000 100000 150000 200000 Número de ciclos De acuerdo al modelo presentado en la ecuación crecimiento de grieta. También, la temperatura es (1), la temperatura está relacionado con el una variable termodinámica que involucra la
|
10.3998/ark.5550190.0012.a07
|
cc-by|cc-by-nc
| null | null |
openalex
|
Novel dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5-triones 5a-j were obtained regioselectively by 1,3-dipolar cycloaddition reaction of 4-arylidene-1-phenylpyrazolidine-3,5diones 2a-e as dipolarophiles with non-stabilized azomethine ylides, generated in situ via decarboxylative condensation of isatins 3a,b and sarcosine 4 in dry ethanol. The prepared compounds were screened for their anti-inflammatory activity "at a dose of 10 mg/kg body weight", especially 5d, 5f, 5h, and 5j which reveal remarkable activities relative to indomethacin which was used as a reference standard in this study.Introduction Cycloadditions are an important group of reactions in organic synthesis. 1 The azomethine ylide represents one of the most reactive and versatile classes of 1,3-dipoles and is trapped readily by a range of dipolarophiles, either inter or intramolecularly, forming substituted pyrrolidines. 2 Spiropyrrolidinyl-oxoindole represents the main alkaloid skeleton of naturally occurring substances such as spirotryprostatine A and spirotryprostatine B which were found to be inhibitors of mammalian cell cycle at G2/M phase, from the secondary metabolites of Aspergillus fumigatus (Figure 1 ). N-Arylpyrazoles are a very interesting class of heterocyclic compounds that have remarkable pharmacological activities in areas such as antibacterialantifungal, 6 hypoglycemic, 7 tumor necrosis inhibition, 8 anti-thrombo-embolic disorders, as antiangiogenic-, 9 and anti-inflammatory effects. 10, 11 In the present work, it is intended to utilize a natural product isatin scaffold for formation of non-stabilized azomethine ylides following the previously described and successful methods, through decarboxylative condensation with α-amino acids and trapping the generated reactive intermediate via 1,3-dipolar cycloaddition reaction with the exocyclic olefinic linkage derived from 1-phenylpyrazolidine-3,5-dione. The anti-inflammatory properties of the prepared compounds will be screened. This work is a continuation of our research directed towards the construction of bio-active spiroheterocycles. Figure 1 . Representative naturally occurring spiropyrrolidinyl-oxoindole alkaloids.
Results and Discussion Chemistry 1-Phenylpyrazolidine-3,5-dione (1) was prepared from malonic acid and phenyl hydrazine, and the different substituted 4-arylidene-1-phenylpyrazolidine-3,5-dione derivatives 2a-e were prepared in accordance with the literature procedure (Scheme 1). 20 Reaction of the 4-arylidene-1-phenylpyrazolidine-3,5-diones 2a-e with non-stabilized azomethine ylides, generated in situ via decarboxylative condensation of sarcosine 4 and isatins 3a,b (Scheme 2) in refluxing ethanol afforded only one product, as indicated by TLC, in a highly regio-and stereo-selective manner (Scheme 3). This cycloaddition is regioselective with the electron-rich carbon of the dipole adding to the β-carbon of the α,β-unsaturated moiety of 2a-e, and is stereoselective, affording only one diastereomer exclusively, despite the presence of three stereo-centers in the product as found in many similar cycloaddition studies. 21, 22 The regioselectivity of the product formation can be explained by considering secondary interaction of the orbitals of the carbonyl group of the dipolarophiles 2a-e with those of the azomethine ylide, as shown in (Figure 2 ). 23 Accordingly, the formation of the observed regioisomers 5a-j via path A is more favorable than 6a-j due to the secondary orbital interaction (SOI) which is not possible in path B. Hence, only one set of regio-isomers 5a-j was formed. The structures of the isolated products were established to be dispiro[pyrazolidine-4,3'pyrrolidine-2',3"-indoline]-2",3,5-triones, 5a-j, rather than the regio-isomeric form dispiro[pyrazolidine-4,4'-pyrrolidine-2',3''-indoline]-2",3,5-triones, 6a-j, based on spectroscopic (IR, 1 H-, 13 C-NMR, MS) and elemental analysis data. 1 H-NMR spectra of 5a-j reveal the presence of three triplet signal sets at δ = 3.54-3.63, 3.95-4.02 ppm (assignable for the magnetically non-equivalent pyrrolidinyl methylene protons H2C-5', coupled with each other and in turn with the vicinal methine proton HC-4') and 4.86-5.01 ppm (corresponding to the pyrrolidinyl methine proton HC-4'), excluding the formation of the other regio-isomeric form 6. If the other isomers 6a-j were formed, one would expect a singlet instead of a triplet for the pyrrolidinyl methylene and methine protons. The 13 C-NMR spectrum of 5b, as a representative example, exhibits the pyrrolidinyl spiro-carbons C-4 (C-3'), C-2' (C-3") at δ = 64.99, 76.33 ppm beside the pyrrolidinyl methine carbon (HC-4') at δ = 45.25, the pyrrolidinyl methylene carbon (H2C-5') appeared at δ = 55.06 ppm. In addition, the indolyl and pyrazolyl carbonyl carbon atoms appeared at δ = 174.67, 174.85 respectively. The mass spectrum of 5b, as a representative example, showed the M + peak at m/z = 372.35 (20 %) and the base peak at m/z = 173.92. Along with the experimental investigation, and for further confirmation of the structures of the synthesized compounds 5a-j, we also carried out some theoretical studies on selected prepared compounds, with the help of molecular modeling software. For example, the calculated heat of formation (∆H) of 5b (Figure 3 ) showed that the most stable form (with the lowest heat of formation) is 2'S, 3'R, 4'R (∆H= 68.622 kcal/mol).
Anti-inflammatory activity The anti-inflammatory activity of the target compounds 5a-j (at a dose of 10 mg/kg body weight) was determined in vivo by the acute carrageenan-induced paw oedema standard. 24 The antiinflammatory properties were recorded at successive time intervals 0.5, 1, 2, 3, and 4h and compared with that of indomethacin (at a dose of 10 mg/kg body weight) which was used as a reference standard. From the obtained results (Table 1 ) it was noticed after 1 h that many of the tested compounds exhibit considerable anti-inflammatory properties (especially 5d, 5f, 5h, and 5j) which reveal remarkable activities, with potency (percentage oedema inhibition of the tested compounds relative to percentage oedema inhibition of indomethacin) of 0.72, 0.64, 0.56, and 0.72 respectively. The structure-activity relationships based on the obtained results indicated that the type of substituents attached to N-1" and C-4' are controlling factors in developing the total pharmacological properties. The best observed anti-inflammatory property is that in which N-1'' bears a methyl group and C-4' is attached to a phenyl group substituted with an electron-donating group (methoxy) in 5j (potency 0.72). However, with an unsubstituted phenyl group, or substitution with electron-withdrawing groups (chlorine and bromine) the function was relatively decreased. It was also noticed that when N-1" is attached to a hydrogen atom, the antiinflammatory activity is increased only when C-4' is attached to a phenyl group substituted with an electron-donating hydroxyl group in 5d (potency 0.72).
Acute toxicity (LD50) The median lethal dose (LD50) of the most active compounds 5d, 5f, 5h, and 5j was determined in mice, according to reported procedures. 25 The results showed that the tested compounds 5d, 5f, 5h, and 5j were non-toxic at doses up to 200 mg / kg.
Experimental Section General. The time required for completion of each reaction was monitored by TLC. All melting points are uncorrected, and were measured on a Gallenkamp apparatus. The IR spectra were recorded on a Shimadzu 470 IR spectrometer (KBr) νmax /cm -1 . The 1 H-, and 13 C-NMR spectra were measured on Varian EM-200 ( 1 H, 200 MHz; 13 C, 50 MHz) spectrometer with TMS as internal standard and DMSO-d6 as solvent. Mass spectra were determined on a JEOL JMS-600 spectrometer. The molecular modeling software is MOPAC (ChemOffice ultra, version 9, 2005). Elemental analyses (C, H, N, and S) were performed on an elemental analysis system GmbH VarioEL V2.3; the results were found to be in good agreement with the calculated values.
Synthesis of dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5-triones (5a-j). General procedure A mixture of the appropriate compounds 2a-e (5 mmol), 3a,b (5 mmol) and sarcosine 4 (0.445 g, 5 mmol) in dry ethanol (20 mL) was boiled under reflux for the appropriate time. Upon completion (monitored by TLC), the reaction mixture was allowed to cool to room temperature. The solid product was filtered off and crystallized from a suitable solvent.
Compound Mean swelling volume ± S.E.M a (% inhibition of oedema)
h 1 h 2 h 3 h 4 h Potency b 5j 0.700 ± 0.040 (8.1) 0.650 ± 0.040 (14.7) 0.625 ± 0.028 (19.3) 0.587 ± 0.025 (23.0) 0.537 ± 0.047 (29.5) 0.72 a S.E.M. = Standard error mean, and all showed at least significant difference at p < 0.05 in comparison with control group. b Potency is expressed as percentage oedema inhibition of the tested compounds relative to percentage oedema inhibition of indomethacin "reference standard" at 1 h effect. Scheme 1 . 1 Scheme 1. Synthesis of 1-phenylpyrazolidine-3,5-dione and 4-Arylidene-1-phenylpyrazolidine-3,5dione derivatives: (a) POCl3, CHCl3, reflux 2h., (b) aromatic aldehydes, glacial AcOH, reflux 30 min.
Scheme 2 . 2 Scheme 2.Mechanism for the generation of azomethine ylide.
4 3aScheme 3 . 43 Scheme 3. Synthesis of dispiro[pyrazolidine-4,3'-pyrrolidine-2',3''-indoline]-2'',3,5-triones.
Figure 2 . 2 Figure 2. Mode of approach of azomethine ylide.
Figure 3 .Figure 3 ( 33 Figure 3. The molecular modeling of 5b.
1 - 1 Phenyl-1'-methyl-4'-(4-bromophenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"indoline]-2",3,5-trione (5c). Reaction time 9 h. Recrystallized from dioxane, m.p. 235-236 C, yield 2.30 g (89 %). IR (KBr): νmax/cm -1 3220 (NH), 3095 (NH), 1725 (C=O), 1695 (C=O), 1615, 1465 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.10 (s, 3H, CH3), 3.51 (t, 1H, upfield H of CH2-CH, J = 18.0 Hz), 3.92 (t, 1H, downfield H of CH2-CH, J = 18.0 Hz), 4.93 (t, 1H, CH-CH2, J = 16.0 Hz), 6.62-7.68 (m, 13H, arom. H), 10.43 (s, 1H, NH), 10.50 (s, 1H, NH); 13 C-NMR (DMSO-d6): δ (ppm) 33.27 (CH3), 45.20 (HC-4'), 56.13 (H2C-5'), 69.89 [spiro C-4 (C-3')], 76.84 [spiro C-2' (C-3'')], 109.72, 118.91, 119.22, 121.63, 123.42, 125.59, 128.35, 128.80, 128.87, 129.46, 130.09, 130.18, 132.13 (arom. CH), 135.11, 135.20, 135.59, 143.13, 143.36 (arom. C), 174.60 (C-2''), 174.84 (C-5), 176.52 (C-3). m/z (%) 518.88 (M + + 2, 33), 516.85 (M + , 45), 354.80 (27), 305.98 (33), 175.02 (100). Anal. Calcd. for C26H21BrN4O3 (517.37): C, 60.36; H, 4.09; Br, 15.44; N, 10.83. Found: C, 60.11; H, 3.85; Br, 15.21; N, 10.68%. 1-Phenyl-1'-methyl-4'-(4-hydroxyphenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"indoline]-2",3,5-trione (5d). Reaction time 12 h. Recrystallized from ethanol; m.p. 243-244 C, yield 1.77 g (78 %). IR (KBr): νmax/cm -1 3250 (br., OH), 3115 (NH), 1715 (C=O), 1700 (C=O), 1695 (C=O), 1610, 1461 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.20 (s, 3H, CH3), 3.59 (t, 1H, upfield H of CH2-CH, J = 16.0 Hz), 4.00 (t, 1H, downfield H of CH2-CH, J = 16.0 Hz), 4.99 (t, 1H, CH-CH2, J = 18.0 Hz), 6.74-7.37 (m, 13H, arom. H), 10.52 (s, 1H, NH), 10.58 (s, 1H, NH), 11.42 (s, 1H, OH); 13 C-NMR (DMSO-d6): δ (ppm) 34.63 (CH3), 45.24 (HC-4'), 55.00 (H2C-5'), 65.20 [spiro C-4 (C-3')], 76.38 [spiro C-2' (C-3'')], 110.
trione (5h). Reaction time 20 h. Recrystallized from ethanol; m.p. 198-199 C, yield 2.10 g (79 %). IR (KBr): νmax/cm -1 3100 (NH), 1710 (C=O), 1700 (C=O), 1690 (C=O), 1610, 1460 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.19 (s, 3H, CH3), 3.16 (s, 3H, CH3), 3.62 (t, 1H, upfield H of CH2-CH, J = 18.0 Hz), 4.00 (t, 1H, downfield H of CH2-CH, J = 18.0 Hz), 4.96 (t, 1H, CH-CH2, J = 16.0 Hz), 6.74-7.77 (m, 13H, arom. H), 10.52 (s, 1H, NH). Anal. Calcd. for C27H23BrN4O3 (531.4): C, 61.03; H, 4.36; Br, 15.04; N, 10.54. Found: C, 60.89; H, 4.44; Br, 14.90; N, 10.36%. 1-Phenyl-1',1"-dimethyl-4'-(4-hydroxyphenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"indoline]-2",3,5-trione (5i). Reaction time 18 h. Recrystallized from ethanol, m.p. 155-156 C, yield 1.64 g (70 %). IR (KBr): νmax/cm -1 3250 (br. OH), 1705 (C=O), 1700 (C=O), 1695 (C=O), 1605, 1460 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.20 (s, 3H, CH3), 2.92 (t, 1H, upfield H of CH2-CH, J = 12.0 Hz), 3.14 (s, 3H, CH3), 3.63 (t, 1H, downfield H of CH2-CH, J = 12.0 Hz), 4.79 (t, 1H, CH-CH2, J = 10.0 Hz), 6.68-7.71 (m, 13H, arom. H), 10.51 (s, 1H, NH), 10.82 (s, 1H, OH); 13 C-NMR (DMSO-d6): δ (ppm) 33.24 (CH3), 39.15 (CH3), 45.56 (HC-4'), 55.14 (H2C-5'), 69.91 [spiro C-4 (C-3')], 76.37 [spiro C-2' (C-3'')], 109.72, 118.79, 119.16, 121.63, 123.42, 125.59, 128.36, 128.80, 128.82, 129.46, 130.00, 130.18, 132.13 (arom. CH), 135.11, 135.31, 135.59, 143.13, 143.33 (arom. C), 174.65 (C-2''), 174.77 (C-5), 176.43 (C-3). Anal. Calcd. for C27H24N4O4 (468.5): C, 69.22; H, 5.16; N, 11.96. Found: C, 69.00; H, 5.02; N, 11.78%. 1-Phenyl-1',1"-dimethyl-4'-(4-methoxyphenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"indoline]-2",3,5-trione (5j). Reaction time 19 h. Recrystallized from ethanol, m.p. 139-141 C, yield 1.81 g (75 %). IR (KBr): νmax/cm -1 3200 (NH), 1705 (C=O), 1700 (C=O), 1605, 1460
Table 1 . 1 In-vivo anti-inflammatory activity of tested compounds using acute carrageenaninduced paw oedema in rats 0 Hz), 1,4'-Diphenyl-1'-methyl-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5-trione (5a). Reaction time 11 h. Recrystallized from dioxane; m.p. 281-282 C, yield 1.99 g (91 %). IR (KBr): νmax/cm -1 3250 (NH), 3100 (NH), 1710 (C=O), 1705 (C=O), 1700 (C=O), 1618, 1460 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.20 (s, 3H, CH3), 3.48 (t, 1H, upfield H of CH2-CH, J = 18.0 Hz), 4.02 (t, 1H, downfield H of CH2-CH, J = 18.0 Hz), 5.00 (t, 1H, CH-CH2, J = 16.0 Hz), 6.71-7.74 (m, 14H, arom. H), 10.50 (s, 1H, NH), 10.55 (s, 1H, NH). Anal. Calcd. for C26H22N4O3 (438.17): C, 71.22; H, 5.06; N, 12.78. Found: C, 71.00; H, 4.91; N, 12.57%. 1-Phenyl-1'-methyl-4'-(4-chlorophenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"indoline] -2",3,5-trione (5b). Reaction time 8 h. Recrystallized from dioxane, m.p. 222-224 C, yield 2.24 g (95 %). IR (KBr): νmax/cm -1 3250 (NH), 3095 (NH), 1725 (C=O), 1695 (C=O), 1616, 1465 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.14 (s, 3H, CH3), 3.54 (t, 1H, upfield H of CH2-CH, J = 14.0 Hz), 3.95 (t, 1H, downfield H of CH2-CH, J = 14.0 Hz), 4.95 (t, 1H, CH-CH2, J = 16.
Table 1 (continued) 1
-methyl-4'-(4-methoxyphenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"- indoline]-2",3,5-trione (5e). Reaction time 10 h. Recrystallized from methanol, m.p. 230-231 C, yield 1.89 g (81 %). IR (KBr): νmax/cm -1 3225 (NH), 3100 (NH), 1715 (C=O), 1700 (C=O), 1610, 1460 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.19 (s, 3H, CH3), 2.97 (t, 1H, upfield H of CH2-CH, J = 14.0 Hz), 3.72 (s, 3H, CH3-O), 3.97 (t, 1H, downfield H of CH2-CH, J = 14.0 Hz), 4.94 (t, 1H, CH-CH2, J = 12.0 Hz), 6.66-7.81 (m, 13H, arom. H), 10.48 (s, 1H, NH), 10.53 (s, 1H, NH). Anal. Calcd. for C27H24N4O4 (468.5): C, 69.22; H, 5.16; N, 11.96. Found: C, 69.05; H, 5.05; N, 11.85%. 1 12, 118.89, 119.16, 121.61, 123.42, 125.59, 128.24, 128.77, 128.87, 129.46, 130.02, 130.18, 132.13 (arom. CH), 135.11, 135.33, 135.69, 143.13, 143.37 (arom. C), 174.67 (C-2''), 174.85 (C-5), 176.44 (C-3). Anal. Calcd. for C26H22N4O4 (454.48): C, 68.71; H, 4.88; N, 12.33. Found: C, 68.41; H, 4.65; N, 12.25%. 1-Phenyl-1'
,4'-Diphenyl-1',1"-dimethyl-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5- trione (5f). Reaction time 18 h. Recrystallized from ethanol, m.p. 181-182 C, yield 1.74 g (77 %). IR (KBr): νmax/cm -1 3100 (NH), 1705 (C=O), 1700 (C=O), 1696 (C=O), 1605, 1460 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.21 (s, 3H, CH3), 2.89 (t, 1H, upfield H of CH2-CH, J = 16.0 Hz), 3.04 (s, 3H, CH3), 3.67 (t, 1H, downfield H of CH2-CH, J = 16.0 Hz), 4.87 (t, 1H, CH-CH2, J = 14.0 Hz), 6.70-7.72 (m, 14H, arom. H), 10.46 (s, 1H, NH). Anal. Calcd. for C27H24N4O3 (452.5): C, 71.67; H, 5.35; N, 12.38. Found: C, 71.51; H, 5.22; N, 12.19%. 1-Phenyl-1
',1"-dimethyl-4'-(4-chlorophenyl)-dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"- indoline]-2",3,5-trione (5g). Reaction time 16 h. Recrystallized from ethanol; mp 191-192 C, yield 1.94 g (80 %). IR (KBr): νmax/cm -1 3200 (NH), 1715 (C=O), 1700 (C=O), 1690 (C=O), 1615, 1465 (C=C); 1 H-NMR (DMSO-d6): δ (ppm) 2.16 (s, 3H, CH3), 3.13 (s, 3H, CH3), 3.65 (t, 1H, upfield H of CH2-CH, J = 18.0 Hz), 4.02 (t, 1H, downfield H of CH2-CH, J = 18.0 Hz), 4.98 (t, 1H, CH-CH2, J = 16.0 Hz), 6.74-7.76 (m, 13H, arom. H), 10.51 (s, 1H, NH). Anal. Calcd. for C27H23ClN4O3 (486.95): C, 66.60; H, 4.76; Cl, 7.28; N, 11.51. Found: C, 66.49; H, 4.57; Cl, 7.10; N, 11.39%.
ARKAT-USA, Inc.
|
10.1002/essoar.10507087.1
|
cc-by
| null | null |
openalex
|
At low wind speeds, the mass flux in saltation is greater than the mass flux of suspended particles (Gordon et al., 2009; Nishimura & Nemoto, 2005) . At high wind speeds, snow transport in suspension becomes relevant and is currently simulated in mesoscale models by advection-diffusion equations (Introduction Wind erosion of snow covered surfaces is frequently observed in alpine and polar regions. Snow transport leads to the formation of bedforms, intensifies snow sublimation and modifies the microstructure of surface snow layers. Moreover, the interaction between the wind field and the complex topography creates regions of enhanced snow erosion and deposition, which greatly contributes to snow height heterogeneity. In alpine regions, these processes are of great importance for water management and avalanche risk assessment (Lehning et al., 2008) . In Antarctica, snow transport is enhanced by the katabatic winds, dominating large areas from the inner plateau to the coast, and clouds of blowing snow particles with a height of hundreds of meters can be observed (Palm et al., 2017) . The aeolian transport of snow occurs at different heights above the ground. The terms drifting snow and blowing snow are commonly used to indicate, respectively, the movement of snow particles close to the surface (up to ∼2 m height) and the movement of smaller snow particles transported at high elevations. Three transport modes (creep, saltation and suspension) are commonly distinguished during snow transport events (Bagnold, 1941) . The rolling and sliding of snow grains along the surface is defined as creep. Creeping particles are typically too large and heavy to be lifted by the flow. During drifting snow events, their motion is mainly driven by impacting particles. Saltation refers to the ballistic motion of particles close to the ground. Particles in saltation generally hit the ground with enough kinetic energy to hop again (rebound) or eject other particles on the bed (splash). They are mostly concentrated in the first 10 cm above the surface and the ensemble of saltating particles constitute the saltation layer. Suspension refers to the motion of smaller snow particles transported above the saltation layer. They mainly follow the wind flow and travel great distances before being deposited on the ground or sublimate.
Flow and Particle Dynamics
Flow Solver The tri-dimensional wind field is solved with the Large Eddy Simulation (LES) technique. Turbulence features larger than the grid size are resolved by the filtered continuity and Navier-Stokes equations, while the effect of smaller eddies is parameterized by a sub-grid scale (SGS) model. The LES model used along with the particles solver is named EPFL-LES. It was developed at the Ecole Polytechnique Fédérale de Lausanne and is based on the work of Albertson and Parlange (1999) . The LES code targets atmospheric boundary layer (ABL) flows, assumed incompressible and driven by a constant streamwise pressure gradient, AA AAAA∞∕AA∂∂[Pa m -1 ] : ∂∂∂∂∞ ∂∂∂∂ = -ρρff uu 2 * LLzz (1) where [kg m -3 ] is the fluid density, [m] is the domain height and * [m s -1 ] is the desired friction velocity. Horizontal gradients are computed with a Fourier-based pseudo-spectral approach and vertical gradients are calculated using second-order finite differences. The time derivatives are computed with the second-order Adams-Bashforth time advancement scheme (Canuto et al., 1988) . In the present code version, the closure SGS model is given by the scale-dependent Lagrangian dynamic model (LASD) as proposed by Bou-Zeid et al. (2005) . This model exhibits better dissipation characteristics than the classic Smagorinsky and the scale-invariant dynamic models. Periodic boundary conditions are imposed in the vertical walls of the computational domain, as required when applying Fourier transforms, allowing for the development of a fully turbulent flow at both the inlet and outlet sections. At the top boundary, impermeability and zero vertical gradients are assumed. At the bottom boundary, the impermeability condition is imposed and the wall shear stress is given by the logarithmic law of the wall. The use of wall functions avoids highly discretized meshes near the surface as well as smaller time steps to guarantee numerical stability. The present LES code has been used in multiple ABL studies concerning land-atmosphere interaction over complex terrains, wind-farms and urban canopy (Albertson & Parlange, 1999; Bou-Zeid et al., 2005; Diebold et al., 2013; Giometto et al., 2016 Giometto et al., , 2017;; Sharma et al., 2017) . A detailed description of the model can be found in these works.
Particle Dynamics Particle motion is computed in a Lagrangian framework. The coupling with the LES solver was developed by Comola (2017) , following the work of Groot Zwaaftink et al. (2014) . The model has been further developed with the contributions of Comola and Lehning (2017) , Sharma et al. (2018) and Comola, Giometto, et al. (2019) . Particle inertia, gravity and aerodynamic drag are related by Newton's second law. Aerodynamic drag, [N] , is given by AA AAii = -1∕2CCAAρρff AAff |UUrr| UUrrrii , where AA AA= 1, 2, 3 denotes the [m] (streamwise), [m] (crosswise) and [m] (vertical) directions in the Cartesian coordinate system. AA AADD is the drag coefficient, [m 2 ] is the particle frontal area, , [m s -1 ] the particle velocity relative to the local flow and | | [m s -1 ] its absolute value (henceforth referred to as AA AArr ). In the current model, saltating particles are assumed spherical, with a frontal area AA AAff = ππππ 2 ∕4 , where [m] is the particle diameter. The drag coefficient is estimated using the expression proposed MELO ET AL.
10.1029/2021JD035260 4 of 26 by Schiller and Nauman (1933) . It is a function of the particle Reynolds number, AA AAAAdd = UUrrdd∕ννff , where [m 2 s -1 ] is the fluid kinematic viscosity: CCDD = 24 RRRRdd ( 1 + 0.15RRRR 0.687 dd ) . (2) This expression was obtained from fitting experimental measurements developed with spherical particles of multiple sizes and is valid for AA AAAAdd < 800 . Hence, it describes both the Stokes and transition flow regimes, which are characteristic of aeolian saltation. The assumption of spherical particles is widely used in saltation models (e.g., Doorschot & Lehning, 2002; Nemoto & Nishimura, 2004; Schmidt, 1980) and in optical measurements of snow size distribution and mass flux (e.g., Crivelli et al., 2016; Guala et al., 2008) . Even though snowfall particles can have multiple shapes according to the meteorological conditions upon formation, particles in saltation exhibit a different shape and size distribution than falling snow: they are generally smaller, denser and more rounded (Nishimura & Nemoto, 2005; Walden et al., 2003; Woods et al., 2008) due to particle fragmentation after multiple impacts with the bed (Comola et al., 2017) . In fact, a layer of wind packed snow composed of small and closely packed grains is commonly observed after drifting snow events (Fierz et al., 2009) . These observations support the assumption of spherical particles when modeling the wind-particle interaction in steady state saltation. However, the drag law could be improved to take into account the effect of surface irregularities (Kok & Renno, 2009) . According to the studies of Dietrich (1982) developed with sand particles, geometrical deviations from a spherical shape can encompass high curvature regions that promote flow separation. Therefore, the drag coefficient is expected to be slightly higher for non-spherical particles. In addition, different drag laws could be employed to better represent fresh snow at saltation onset or when drifting snow occurs with concurrent snowfall, as proposed by Tagliavini et al. (2021) for falling snow crystals. The equation for particle trajectory yields: ddddppppp = [ 3 4 ρρff ρρpp CCDD dd UUrr (ddpp -ddppppp) -ggggpp3 ] dddd (3) where , [m s -1 ] is the particle velocity, [m s -1 ] is the instantaneous flow velocity resolved by the LES solver, [kg m -3 ] is the particle density, [m s -2 ] is the acceleration of gravity, [s] is the time variable and AA AA is the Kronecker delta. Equation 3 is solved numerically with a first-order forward Euler method. Other forces such as aerodynamic lift, electrostatic forces and those from interparticle collision are expected to be smaller than weight and drag and are generally neglected when modeling saltation in air (Anderson & Hallet, 1986; Maxey & Riley, 1983) . Their effect on sand saltation was studied by several authors (e.g., Durán et al., 2011; Huang et al., 2007; Kok & Renno, 2006 , 2008; Schmidt et al., 1998) and further investigation is needed to fully assess their impact on particle trajectory (Kok et al., 2012) . In previous works based on this model (Comola, Giometto, et al., 2019; Groot Zwaaftink et al., 2014; Sharma et al., 2018) , the non-resolved SGS velocities were computed. Then, the instantaneous wind field was derived from the sum of the resolved wind velocity field, AA AAii , and the SGS velocities. The modeling of velocity fluctuations is important when using simple flow models, as COMSALT (Kok & Renno, 2009) , or RANS solvers (Nemoto & Nishimura, 2004) . In these models, turbulence is not resolved and a model for high-frequency velocity fluctuations is imperative. However, the importance of such a model is less clear for LES, as the large scale instantaneous turbulent flow is provided as a solution of the flow solver. In fact, Dupont et al. (2013) concluded that the SGS velocities have a negligible effect on particle trajectories. Moreover, Z. Wang et al. (2019) did not consider the SGS velocities when modeling saltation with an LES solver. The impact of SGS velocities on particle trajectories may also depend on the SGS model employed, even though there are no works in the literature regarding this question. In this work, the effect of the SGS turbulence features on the resolved wind velocity field is modeled with one of the most advanced SGS closure schemes, the LASD (Bou-Zeid et al., 2005) . Thus, the effect of the SGS velocities on particle motion is assumed to be negligible and not taken into account. The effect of snow sublimation is also neglected in this study. This simplifies the analysis and avoids the computational cost of solving the thermodynamic interaction between the particles and the air. Recent studies have shown that snow sublimation can be significant in the saltation layer, despite the high values of relative humidity MELO ET AL. 10.1029/2021JD035260 5 of 26 (Sigmund et al., 2021; Z. Wang et al., 2019) . Therefore, the modeling of particle sublimation is particularly important when assessing heat and water vapor transport in the atmosphere during drifting snow events. However, this is outside the scope of this study. The effect of snow sublimation on particle dynamics itself is restricted to a reduction in particles size and to an increase in particles sphericity. The feedback of particle motion on flow momentum is modeled through a source term, [N m -3 ] , in the Navier-Stokes equations. AA AAii is given by the total drag force induced by the particles, corresponding to the sum of AA -DDii , per unit volume. The contribution of each particle is linearly extrapolated to the nearest eight grid nodes where LES is resolved. Periodic boundary conditions are applied to particles exiting the domain through its vertical walls. Particles that reach the top boundary are assumed to leave the domain and those impacting the bottom boundary (erodible bed) may rebound and eject other grains as described in Section 2.3. Different studies have been conducted with previous and current versions of this model concerning snow saltation variability (Groot Zwaaftink et al., 2014) , drifting snow sublimation (Sharma et al., 2018; Sigmund et al., 2021) and preferential deposition over hills (Comola, Giometto, et al., 2019) . A detailed description of the model algorithm and a comparison between simulation results and field/wind tunnel measurements can be found in these works.
Surface Processes The interaction between surface grains, the wind flow and particles impacting the bed is described by three main processes: aerodynamic entrainment, rebound and splash. These surface processes are modeled with statistical models based on physical principles and experimental correlations, as proposed by Groot Zwaaftink et al. ( 2014 ) and further developed by Comola and Lehning (2017) . This approach reduces the computational cost associated with the direct numerical simulation of particle interactions within the granular bed. Saltation models based on the Discrete Element Method (DEM) simulate these complex interactions, but are not suitable for simulating particle transport over large computational domains (Comola, Gaume, et al., 2019; Durán et al., 2012; Pähtz et al., 2015) .
Aerodynamic Entrainment When a fluid flows over a granular and erodible bed, surface particles can be moved and eventually lifted by the flow. This process is called aerodynamic entrainment and occurs when the fluid surface shear stress grows above a given threshold. This threshold, that defines the start of wind erosion, is estimated by considering the forces applied on a grain laying on the bed and by performing a balance of angular momentum. The quantity of interest is the minimum aerodynamic force that makes the grain rotate over its leeward point of contact with the underlying grains and, eventually, leads to an uplift of the grain. In general, this threshold shear stress is modeled as a mean quantity, related to the instantaneous aerodynamic force by a parameterization. Bagnold (1941) named it the fluid threshold, [Pa] . Considering particle weight, buoyancy and drag, he proposed the following well known expression: ττffff = AA 2 (ρρpp -ρρff ) gggg (4) where AA AA is the fluid threshold coefficient, which depends on different flow and particle characteristics. Chepil (1959) deduced an expression for AA AA , function of the turbulence intensity, particle geometry and drag coefficient, estimated by a series of experiments developed with sand and soil grains. Bagnold (1941) proposed AA AA= 0.1 for sand beds, after a series of wind tunnel and field experiments. A higher value is expected for very small particles like dust. In this case, the granular surface is not aerodynamically rough and a thin viscous sub-layer is present close to the surface, which limits the transport of flow momentum to the bed. Different criteria have been proposed to define the onset of aerodynamic entrainment. A summary of the latest developments can be found in Pähtz et al. (2020) . Interparticle forces, as the van der Waals and electrostatic forces and those induced by interparticle bonds, also play a role in the aerodynamic entrainment of cohesive materials as snow or moist soils (Schmidt, 1980; Shao & Lu, 2000) . However, the quantification of such forces is still a challenge. The contribution of interparticle ice bonds MELO ET AL. 10.1029/2021JD035260 6 of 26 in the calculation of the fluid threshold is of special interest when studying the erosion of snow covered surfaces and was firstly addressed by Schmidt (1980) . However, for common interparticle bond radius, the values estimated for AA AAffff were too large for pure aerodynamic entrainment of snow particles to occur. Other authors, as Lehning et al. (2000) and Clifton et al. (2006) , used the same approach suggested by Schmidt (1980) , but adjusted the bond properties and empirical constants to improve the agreement with wind tunnel tests performed with natural snow beds. The values for AA AAffff obtained during wind tunnel and field experiments are lower than those deduced by Schmidt (1980) , possibly because patches of loose snow grains are always present over dry snow surfaces. These grains can be easily lifted by the flow and contribute to the development of saltation by further ejecting other particles. Moreover, bed microtopography can also induce local peaks in shear stress, leading to the preferential entrainment of grains more exposed to the airflow. The contribution of interparticle cohesion to the fluid threshold can also be taken into account by adjusting the value of the fluid threshold coefficient. For example, Clifton et al. (2006) proposed a value of A = 0.18 from fitting Equation 4 to wind tunnel measurements developed with different snow surfaces. In light of the challenges and uncertainties to correctly quantify the effect of interparticle forces on the fluid threshold, these forces are not taken into account in the calculation of AA AAffff . Hence, AA AAffff is computed from Equation 4, considering the grain mean diameter, ⟨ ⟩ [m] , and a constant value for AA AA equal to 0.1 as proposed by Bagnold (1941) . This is a simpler approach suitable to study steady state saltation, where the contribution of aerodynamic entrainment is expected to be negligible in comparison to rebound/splash entrainment (Kok et al., 2012) . Indeed, for non-cohesive materials like sand, it is well known that the surface shear stress, [Pa] , stays below the fluid threshold during saltation events, which strongly reduces the occurrence of aerodynamic entrainment (e.g., Bagnold, 1941; Owen, 1964) . AA AAss is related to the surface friction velocity, * , [m s -1 ] , by its definition: AA AAss = ρρff uu 2 * ,ss . The surface friction velocity differs from the imposed friction velocity, AA AA * , after saltation onset and the consequent exchange of momentum from the fluid to the particles. For cohesive material like snow, wind tunnel measurements performed by Paterna et al. (2017) have also shown the predominance of rebound/splash mechanisms over aerodynamic entrainment when the wind strength is sufficiently strong for steady state saltation to develop. Naturally, when studying intermittent saltation, a more accurate description of the fluid threshold is required. The number of grains entrained per unit area per unit time, defined as the aerodynamic entrainment rate, [m -2 s -1 ] , is computed using the expression proposed by Anderson and Haff (1991) : NNaaaa = ηη (ττss -ττffff) (5) where AA AA[N -1 s -1 ] is the entrainment coefficient, computed with the expression proposed by Doorschot and Lehning (2002) : ηη = CCaaaa 8ππ⟨dd⟩ 2 (6) where the coefficient Groot Zwaaftink et al., 2014) . As we are interested in modeling transport limited saltation -a saltation regime for which the amount of saltating particles is only limited by the availability of wind momentum (Kok et al., 2012) -the initial particle concentration at the surface is considered high enough so that there is never a shortage in the supply of erodible particles. However, this is not always the case in snow covered regions, where thin erodible snow layers can exist on top of hard and sintered snow. [m 2 N -1 s -1 ] is set to 1.5 AA m 2 N -1 s -1 ( In the model, entrained particles start their trajectory at a height of four times the mean grain diameter. The initial velocity and vertical angle of ejection are defined according to a lognormal distribution as described in Clifton and Lehning (2008) . The mean and standard deviation of the distribution are computed with the expressions presented in Table 1 . The horizontal angle of ejection is given by the horizontal flow direction.
Rebound After impacting the surface, a grain may rebound and eject other particles laying on the bed. The probability of rebound, AA AArr , is described by the expression proposed by Anderson and Haff (1991) : PPrr = PPmm [ 1 -exp (-γγ|uuppppp |) ] (7) MELO ET AL. ] is given by AA |uuppppp| = √ εεrr|uuppppp | , where AA AArr is the fraction of kinetic energy retained by the rebounding grain (restitution coefficient). Saltation models have shown to be highly sensitive to the value of AA AArr , which greatly depends on the particle elastic properties (Kok & Renno, 2009) . Experiments developed with sand showed that AA AArr varies according to a normal distribution (D. Wang et al., 2008) . Although the restitution coefficient for snow particles is more uncertain, experiments have not suggested a significant deviation from the values obtained for sand grains (Nalpanis et al., 1993) . The horizontal angle of rebound is given by the horizontal flow direction and the vertical angle is computed from an exponential distribution. Further details are presented in Table 1 .
Splash When a grain impacts the bed, it can eject several grains initially at rest. This process, named splash or ejection, is the main driver of particle motion during steady state saltation (Kok et al., 2012; Paterna et al., 2017) . As flow momentum decreases near the surface due to particle drag, aerodynamic entrainment is highly compromised after the start of saltation. Particles impacting the ground become the main source of momentum as they travel from high momentum regions to the surface. Numerous statistical splash functions have been proposed to estimate the number of ejected grains, AA AA , and their initial velocity, | , | [m s -1 ] , as a function of the impacting grain velocity, AA |uuppppp | , and mass, [kg] (e.g., Anderson & Haff, 1988; McEwan & Willetts, 1991) . In this work, the number of ejected grains is computed from energy and momentum conservation laws, as proposed by Kok and Renno (2009) and adapted by Comola and Lehning (2017) to take into account the effect of mixed-sized grains and interparticle cohesion. The impacting grain and the bed are regarded as an isolated system, for which energy and momentum conservation is applied. A fraction of the kinetic energy and momentum, AA AArr and AA AArr , respectively, is kept by the impacting grain leading to its rebound. The remaining fraction is only partly transferred to the ejected grains, as a fraction of the impacting energy and momentum, AA AAff and AA AAff , respectively, leads to the rearrangement of surface grains and, consequently, to friction related losses. Vertical angle (a) Lognormal AA 75 -55 [ 1 -exp ( -dd 175×10 -6 )] 15 Clifton and Lehning ( 2008 )
Rebound Velocity magnitude Kok and Renno (2009) Vertical angle (a) Exponential 45 -Kok and Renno ( 2009 ) - AA √ εεrr | | uuppppp | | -
Splash Velocity magnitude Exponential AA 0.25 | | uuppppp | | 0.3 - Sharma et al. ( 2018 ) Vertical angle (a) Exponential 50 - Rice et al. (1995 Rice et al. ( , 1996) ) Horizontal angle (b) Normal 0 15 Xing and He ( 2013 ) Note. Velocities are in units of AA m s -1 , angles are in degrees and the grain size is in meters. (a) Measured from a horizontal plane (downwind between 0 and 90 ). (b) Measured from the plane of particle impact. (Bagnold, 1941) . Comola and Lehning (2017) arrived to the following expressions: NNEE = (1 -PPrrεεrr -εεff ) mmII uu 2 pppII ⟨mm⟩⟨uu 2 ppppp ⟩ + rrEEσσmmσσ uu 2 ppppp + 2φφ (8a) NNMM = (1 -PPrrμμrr -μμff ) mmII uupppII cosααII ⟨mm⟩⟨uuppppp⟩⟨cosαα⟩⟨cosββ⟩ + rrMM σσmmσσuu ppppp (8b) where AA AAEE and AA AAMM denote the number of ejected grains computed by the energy and momentum equations, respectively. The quantities within angle brackets represent average values, [kg] being the mass of an ejected grain, [ ] the vertical angle of ejection and [ ] the horizontal angle of ejection measured from the plane of impact (in the above equations, both AA AA and AA AA are assumed statistically independent). [kg] , , [m s -1 ] and 2 , [m 2 s -2 ] denote the standard deviation of AA AA , AA AAppppp and AA AA 2 ppppp , respectively. ppppp and between AA AA and AA AAppppp , respectively, and [J] is the energy required to break the cohesive bonds between each ejected grain and the surrounding ones. The modulus symbol in both AA AAppppp and AA AAppppp was suppressed for simplicity. The number of ejected grains is then given by the minimum value between AA AAEE and AA AAMM , which guarantees that neither energy nor momentum is created. The number of ejected grains is expected to be restricted by momentum conservation when the bed is constituted by loose grains (Kok & Renno, 2009) . However, this is not always obtained when interparticle forces are present (Comola & Lehning, 2017; Shao et al., 1993) . The main difference between the splash dynamics of sand and snow particles lies indeed in the nature of the interparticle forces (Comola & Lehning, 2017) . In snow, they arise from ice bonds among neighboring particle, while in sand they are caused by the occasional presence of water menisci. Several parameters in the splash functions are expected to depend on the material properties, such as the ejection velocity of splashed grains and the restitution coefficient, AA AArr . In the particular case of snow, metamorphic changes in the snow surface may also induce variations in the parameters. Moreover, in the work of Doorschot et al. (2004) , the authors question the occurrence of rebound and splash for fresh snow. Despite these differences, the studies of Nalpanis et al. (1993) and Nishimura and Hunt (2000) have shown some similarities between the main snow and sand splash parameters. The parameters' values considered in this study are therefore mainly based on sand experiments (e.g., Anderson & Haff, 1988 , 1991; Kok & Renno, 2009; Rice et al., 1995 Rice et al., , 1996;; Xing & He, 2013) . The velocity and angle of ejection are defined according to specific probability distributions (Table 1 ) and the parameters AA AArr, AAff , μμrr, μμff , rrEE, rrMM , ⟨cosαα⟩ and AA ⟨cosββ⟩ are assumed constant (Table 2 ). Sensitivity analysis carried out by Comola and Lehning (2017) revealed that the splash model used in this study is robust to variations of up to AA ± 20% in the model parameters. Additional studies on the splash mechanics of natural snow (see e.g., Araoka & Maeno, 1981; Nishimura & Hunt, 2000) would ultimately help reducing the model uncertainties. 2013 ) φ [J] (a) AA 10 -10 , 5 × 10 -10 , 5 × 10 -9 Gauer (2001) (a) Values obtained for ice particles. AA AA= 0 J is considered for loose grains. The correlation coefficients, AA AAEE and AA AAMM , are set to zero, as in Comola and Lehning (2017) . The mean and standard deviation of the mass of ejected grains are computed assuming equally sized grains or a lognormal distribution for the grain diameter (Colbeck, 1986) . Finally, the cohesion energy, AA AA , is set to different figures throughout the simulations according to the range proposed by Gauer (2001) and investigated by Comola and Lehning (2017) .
Numerical Setup
General Settings The computational domain is a cube of 6.4 m side length. It models the near surface atmospheric flow over a flat erodible bed. The domain is relatively short in both horizontal directions, especially in the streamwise one. This is partially compensated by applying periodic boundary conditions. However, the use of a longer domain is necessary for the consistent development of large coherent structures observed in experimental and numerical boundary layer studies (Munters et al., 2016) . Even though longer domains are imperative for a proper comparison with experimental data, a cubic domain was considered adequate for the study of steady state saltation developed in this paper. Moreover, it greatly reduces the computational time. The domain is discretized in 64 cells of equal size in the streamwise and crosswise directions. The vertical direction is discretized in 128 cells using a hyperbolic function. The hyperbolic function guarantees a more refined mesh close to the bottom boundary, with an approximately constant thickness of 1 cm in the first 15 cm. The first grid center point is placed in the logarithmic sublayer, at ∼0.5 cm height. The simulations are performed over a total of 350 s to allow the development of steady state saltation. The time step is set to AA 5 × 10 -5 s for both the flow and particle solvers. The flow is allowed to develop over 25 s prior to the start of surface erosion. The initial streamwise component of the velocity field is given by a logarithmic profile, function of AA AA * and of the roughness length, [m] . The roughness length is assumed constant along the surface and equal to AA 10 -5 m. The initial crosswise and vertical velocity components are set to zero. White noise is added to all initial velocity components to accelerate the development of a fully developed turbulent flow. The fluid density and kinematic viscosity are set to AA AAff = 1.34 kg m -3 and AA AAff = 1.24 × 10 -5 m 2 s -1 , respectively. Particles are modeled as ice spheres with density AA AApp = 918.4 kg m -3 . The top of the erodible surface is defined at a height AA AA= 0 m and particle size is assumed uniform or defined by a lognormal distribution, characterized by the grain mean diameter, AA ⟨dd⟩ , and standard deviation, [m] . In order to reduce the computational cost of the simulations, particles are not modeled individually but grouped in parcels, constituted by particles of equal size that follow the same trajectory. Particles from the same parcel were aerodynamically entrained at the same surface location and time step, or were ejected from the same impact event. The number of particles per parcel can assume a value between 5,000 and 250,000. As a consequence, the number of parcels aloft varies from 5,000 to 20,000 during steady state conditions, for friction velocities ranging from 0.4 to 0.8 AA m s -1 . This assumption is considered reasonable for the analysis of time-averaged quantities performed in this work.
Simulation Details In order to study the effect of friction velocity, mean grain size, size distribution and cohesion energy on saltation dynamics, four groups of simulations are performed -S1 to S4 -for which different values of AA AA * , AA ⟨dd⟩ , AA AAdd and AA AA are considered. The parameters used in each simulation group are summarized in Table 3 . In simulations S1 and S2, a bed of equally sized ( AA AAdd = 0 μμm ) and loose grains ( AA AA= 0 J ) is modeled. In S1, the effect of the imposed friction velocity is studied while keeping the remaining parameters unchanged. In S2, different values for the grain diameter, AA ⟨dd⟩ , are tested. In simulations S3 and S4, a bed of mixed-sized grains is modeled by describing the grain size by a lognormal distribution. In S3, the effect of the standard deviation of the distribution, AA AAdd , on steady state saltation is analyzed. Finally, in S4, interparticle forces are assumed between surface grains and different values for the cohesion energy,
Data Post-Processing The vertical profiles of particle concentration, mean particle streamwise velocity and particle mass flux are computed by dividing the computational domain in horizontal layers of thickness AA Δzzkk[m] . The particle concentration, [kg m -3 ] , is given by cc(zzkk) = ∑ NN kk nn=1 mmnn LLxxLLyyΔzzkk (9) where AA AAkk is the number of particles in the horizontal layer with mean height [m] , [kg] is the mass of the AA AA ttt particle, [m] is the domain length and [m] is the domain width. The mean particle velocity in the streamwise direction, ⟨ ,1⟩ [m s -1 ] , is given by the arithmetic mean. The particle mass flux, [kg m -2 s -1 ] , is given by the product of the particle concentration and the mass-weighted average particle streamwise velocity, yielding qq(zzkk) = ∑ NN kk nn=1 mmnnuuppp1 nn LLxxLLyyΔzzkk (10) where ,1 [m s -1 ] is the streamwise velocity of the AA AA ttt particle in layer AA AA . The integrated mass flux of saltating particles, [kg m -1 s -1 ] is computed by integrating particle mass flux, AA AA , along the height, from the surface to 15 cm. The last 100 s of each simulation are used to compute the time-averaged values of AA AA , AA ⟨uuppp1⟩ , AA AA and AA AA . During this time interval (250-350 s), the changes in total mass of particles aloft are negligible and saltation is assumed to be in steady state. The surface friction velocity, AA AA * ,ss , at each time step is obtained by averaging over the surface. The time-averaged value obtained for the last 100 s of each simulation is defined as the equilibrium surface friction velocity, * , [m s -1 ] .
Results and Discussion In this section, the results are presented and discussed. Results obtained with simulations S1 to S4 are analyzed in Sections 4.1 to 4.4, respectively. Moreover, a comparison with existing saltation models and with the conclusions drawn from the latest wind tunnel and field experiments is presented.
The Effect of Friction Velocity In simulations S1, a bed of equally sized and loose grains with a diameter of 200 AA AAm is modeled. The streamwise wind speed profiles are presented in Figure 1 . They are computed by averaging the streamwise velocity along horizontal planes. The profiles are time-averaged over the first 25 s and over the last 100 s of each simulation (before saltation onset and during steady state saltation). As expected, the resulting wind speed is lower for the latter, as the saltation layer acts on the flow as an additional sink of momentum. The velocity profiles obtained during steady state saltation intercept in a point at ∼7 mm above the surface (inset in Figure 1 ). This characteristic feature of steady state saltation was originally observed by Bagnold (1941) when performing wind tunnel experiments. This point is defined as the focus point. Previous saltation models have characterized the wind velocity profile in the saltation layer using a logarithmic profile and have assumed the existence of a focus point (e.g., Pomeroy & Gray, 1990) . This greatly simplifies the description of the velocity field in the saltation layer and, consequently, the theoretical modeling of saltation. Numerical models based on parameterizations of splash entrainment have also reproduced this characteristic feature (Kok et al., 2012) . The height of the focus point is expected to vary between 1 and 10 mm (Bagnold, 1941; Kok et al., 2012) . In order to accurately assess its location, a more refined mesh near the surface would have to be employed. An equivalent surface roughness, characteristic of each saltation layer, can be estimated from the velocity profiles obtained during steady state saltation (Dupont et al., 2013) . By extending the velocity profiles down to the wall, zero velocity is attained at greater heights as AA AA * increases. Hence, the equivalent surface roughness increases with AA AA * . This is related to an enhanced momentum exchange between the fluid flow and the particles aloft when AA AA * increases. Therefore, it is ultimately related to the increase in particle mass flux. The time-averaged vertical profiles of particle mass flux, concentration and mean streamwise velocity are presented in Figures 2a-2c . The average is performed over the last 100 s of each simulation. Particle mass flux decreases with height and increases with AA AA * (Figure 2a ), as previously observed in field measurements (Nishimura et al., 2014) . A similar trend is observed for particle concentration (Figure 2b ). Several saltation models have assumed an exponential decay for the vertical profile of particle mass flux of the form AA AA(zz) = AAmmexp(-zz∕llrr) , where [m] is a reference height, commonly related to the height of the saltation layer, and [kg m -2 s -1 ] is given by the ratio of the integrated mass flux, AA AA , to the reference height, AA AArr (Clifton et al., 2006; Martin & Kok, 2017; Nishimura & Hunt, 2000; Vionnet et al., 2014) . Some saltation models (Clifton et al., 2006; Vionnet et al., 2014) define AA AArr = uu 2 * ∕(λλλλ) , where AA AA= 0.45 , as proposed by Nishimura and Hunt (2000) after wind tunnel experiments developed with fine-grained natural snow. We present the vertical profiles of particle mass flux in a semi-logarithmic scale in Figure 3 . The fitting of the simulation results to an exponential decay is also presented. The fit is performed up to a height of 3, 5 and 14 cm for AA AA * equal to 0.4, 0.6 and 0.8 AA m s -1 , respectively. Moreover, the near surface value of AA AA is neglected in the fit. The reference height, AA AArr , obtained from the fit and from the expression proposed by Nishimura and Hunt (2000) is also indicated at each exponential curve. It can be seen that particle mass flux decreases exponentially with height in the first centimeters above the ground. Moreover, a slight increase of AA AArr with AA AA * is obtained, which implies a weak variation of the saltation layer height with AA AA * . This trend was also obtained by Nalpanis et al. (1993) and Martin and Kok (2017) after wind tunnel and field experiments, respectively, developed with sand. However, it contrasts with the stronger increase proposed by Nishimura and Hunt (2000) . Simpler saltation models assume a constant particle mass flux in the saltation layer, ⟨ ⟩ [kg m -2 s -1 ] , so that AA AA= ⟨qq⟩hss , where h [m] is the height of the saltation layer (Pomeroy & Gray, 1990) . Particle mass flux is given by the product of particle concentration and particle streamwise velocity, as described in Section 3.3. Particle concentration at the saltation layer height is the quantity of interest when modeling snow suspension (e.g., Amory et al., 2015 Amory et al., , 2021;; Lenaerts et al., 2012; Vionnet et al., 2014) . In order to compute it, particle mass flux at AA Ass must be known. Taking into account the strong variation of AA AA with height (Figure 3 ), the assumption of a mean value will hardly deliver a reasonable estimate of AA AA(hss) , even if an accurate description for AA AA and AA Ass is considered. Different criteria have been proposed to define the height of the saltation layer. They are either based on the mean height of particle trajectories (Owen, 1964) , the amount of momentum transported from the fluid to the particles (Dupont et al., 2013) , or the exponential fit of the mass flux profile (Martin & Kok, 2017) . Due to the lack of consensus in this matter, a precise definition of the saltation layer height is avoided in this work. Particle streamwise velocity increases with height and AA AA * (and, therefore, with wind speed), as shown in Figure 2c . For heights smaller than 1 cm (approximately), the variation of particle streamwise velocity with AA AA * is negligible (inset in Figure 2c ). This is predicted by existing saltation models (Kok & Renno, 2009) and wind tunnel measurements (Ho et al., 2011) . This result is also obtained theoretically, based on the notion that steady state saltation is characterized by a mean replacement capacity equal to one (Kok et al., 2012) . This means that, on average, one grain enters the saltation layer each time an impacting grain fails to rebound. Assuming that saltation is mainly dominated by splash, this condition is met for a given impact velocity, which completely defines the number of ejected grains and the probability of rebound for a given bed type (see Equations 7 and 8). Hence, it follows that the particle speed near the surface is independent of AA AA * and rather varies with the bed characteristics. The near surface particle speed is closely linked to the focus point (or Bagnold's focus) observed in the average streamwise wind speed profiles (Figure 1 ). Saltating particles are accelerated by the flow along their trajectories, therefore, the near surface particle speed can only be approximately invariant with regards to AA AA * if the near surface wind speed is also approximately invariant with regards to the same quantity. High above the surface, the wind speed increases as AA AA * rises. Hence, a near surface wind speed approximately invariant with AA AA * is only obtained if a focus point is visible close to the surface, below which the wind speed decreases as AA AA * increases. Some of the snow saltation models implemented in atmospheric models (Pomeroy & Gray, 1990; Vionnet et al., 2014 ) assume that the mean particle streamwise velocity in the saltation layer is invariant with AA AA * . In addition, they assume that the saltation layer height varies from 1 to 5 cm (or from 2 to 6 cm) for AA AA * varying from 0.4 to 0.8 AA m s -1 , respectively. Indeed, our results show that the mean particle speed does not vary with AA AA * in the first centimeter above the ground (inset in Figure 2c ). However, this is not considered representative of the whole saltation layer. The wind tunnel measurements of Ho et al. (2011) revealed a negligible increase of particle speed with AA AA * up to a height of 4 cm. However, the field measurements of Nishimura et al. (2014) showed a clear increase of particle speed with friction velocity between 1 and 10 cm height. In large scale models, the correct estimate of particle speed is required for the calculation of particle concentration, which is used as a lower boundary condition for suspension. The surface friction velocity, AA AA * ,ss , as a function of time is presented in Figure 4 . The fluid threshold friction velocity, * , [m s -1 ] , related to the fluid threshold shear stress by AA AAffff = ρρff uu 2 * ,ff ff , is also plotted as a reference. AA AA * ,ss strongly decreases immediately after the start of surface erosion ( AA AA= 25 s). It tends to an equilibrium value -the equilibrium surface friction velocity, AA AA * ,eeee . A small reduction of AA AA * ,eeee is obtained when the imposed friction velocity, AA AA * , increases (inset in Figure 4 ). The numerical model COMSALT proposed by Kok and Renno (2009) also predicts this trend for a bed with uniform grain size (Kok et al., 2012) . However, they predicted a stronger reduction than that presented in the inset in Figure 4 . The wind tunnel experiments performed by Walter et al. (2014) revealed a non-monotonic evolution of AA AA * ,eeee with AA AA * . During the experiments, AA AA * was continuously increased above the fluid threshold friction velocity. As a result, the measured AA AA * ,eeee firstly reduced and then increased. In general, a relatively small variation of AA AA * ,eeee with AA AA * and a relatively large standard deviation of the measurements were obtained, 2014 ) considered the assumption of a constant surface friction velocity (function of the grain type but invariant with the wind speed, as proposed by Owen (1964) ) a reasonable first-order approximation. The impact threshold friction velocity, * , [m s -1 ] , is generally defined as the minimum friction velocity, AA AA * , at which saltation can be sustained after its onset (Bagnold, 1941) . In the work of Kok and Renno (2009) , the impact threshold friction velocity is assumed equal to the minimum value of AA AA * that satisfies the steady state equation. In their model, the equilibrium friction velocity, AA AA * ,eeee , tends to the computed AA AA * ,iiii as AA AA * decreases (Kok et al., 2012) . Taking into account these results, a simplified approach is followed in this work and AA AA * ,iiii is given by the value of AA AA * ,eeee obtained when AA AA * is set to 0.4 AA m s -1 (the minimum friction velocity common to all simulation groups). This approach is considered appropriate taking into account the small variation of AA AA * ,eeee with AA AA * obtained for most simulations. A more accurate estimation of the impact threshold friction velocity needs further investigation, in particular, a set of simulations at low friction velocities (near the impact and fluid threshold friction velocities) and the analysis of the transition from intermittent to steady state saltation. The mass of particles aloft per unit surface area varies with time, as presented in Figure 5 . The vertical mass flux of particles leaving the surface either through aerodynamic entrainment or splash and the vertical mass flux of . A longer simulation time of 600 s was considered in this case to better illustrate the steady state regime. The evolution shown in the first 350 s is representative of all the simulations performed. At AA AA= 25 s, saltation starts due to aerodynamic entrainment. A sudden increase in the mass of particles aloft is observed, which is consistent with the strong decrease in surface friction velocity presented in Figure 4 . The overshoot in particle mass is justified by the surge in the vertical mass flux of particles entering saltation via splash, that overcomes the vertical mass flux of particles leaving the saltation layer through deposition (Figure 5b ). The imbalance between the vertical mass flux of splash and deposition drives the variation of mass of particles aloft. When saltation reaches steady state, a dynamic equilibrium between the vertical mass flux of splash and deposition is obtained. Aerodynamic entrainment is much smaller than splash: the vertical mass flux reaches a maximum at saltation onset and then decreases to a steady state value, which is one order of magnitude lower than the vertical mass flux of splash and deposition. In the simulations performed, aerodynamic entrainment occurs during steady state saltation because the surface friction velocity is greater than the specified fluid threshold friction velocity (Figure 4 ). However, taking into account the relatively small contribution of aerodynamic entrainment to the mass of particles aloft, the correct assessment of the fluid threshold is expected to have a negligible effect on steady state saltation for friction velocities significantly greater than the fluid threshold friction velocity. These results are in agreement with the notion that steady state saltation is dominated by splash and that an equilibrium between splash and failure of rebound should be attained (Kok et al., 2012; Paterna et al., 2017) . The time-averaged integrated mass flux and the corresponding standard deviation are presented in Figure 6 . In Figure 6a , the fit between the mean values and a quadratic function is presented, as well as between the mean values and a cubic function. Moreover, the integrated mass flux estimated from fitting the vertical profile of particle mass flux to an exponential function is also shown. In Figure 6b , the results are compared to saltation models proposed by several authors (Bagnold, 1941; Doorschot & Lehning, 2002; Durán et al., 2011; Pomeroy & Gray, 1990; Sørensen, 2004) . The results from Doorschot and Lehning (2002) were obtained from the numerical algorithm proposed by the authors. The remaining curves are computed from the equations presented in Table 4 . Equations used to compute the integrated mass flux (as those presented in Table 4 ) are obtained from the balance of horizontal momentum applied to the saltating particles. The total horizontal force per unit area applied on these particles is equal to the excess shear stress, AA AA-AAss = ρρff ( uu 2 * -uu 2 * ,ss ) , where is the surface shear stress before saltation onset ( AA AA = ρρff uu 2 * ). In addition, if particle trajectories are characterized by a representative hop, with length [m] , in which particles undergo a mean variation of horizontal velocity, Δ h [m s -1 ] , lift off and impact with the bed, the integrated mass flux is computed from AA AA= ρρff ( uu 2 * -uu 2 * ,ss ) LL∕Δuupp h (e.g., Kok et al., 2012) . Different models arise from different assumptions regarding the evolution of AA AA * ,ss and AA AA∕Δuupp h . Following Owen's hypothesis (Owen, 1964) , the surface friction velocity, AA AA * ,ss , is generally assumed invariant with respect to AA AA * and equal to the impact threshold friction velocity, AA AA * ,iiii . Even though there is no full consensus on the validity of this hypothesis and its implications on saltation dynamics (see, for instance, Kok et al. (2012) and Walter et al. (2014) ), the fact that the general equation yields AA AA= 0 when AA AA * equals AA AA * ,ss favors the use of this simplifying assumption. The quadratic growth of AA AA with AA AA * is predicted theoretically when both the particle velocity near the surface (and, consequently, AA Δuupp h ) and the representative hop length are considered invariant with AA AA * (Durán et al., 2011; Ungar & Haff, 1987) . This yields an expression for AA AA of the form AA AAAA 2 * + bb , which is corroborated by recent field experiments (e.g., Martin & Kok, 2017) . The increase of AA AA with AA AA 3 * was early proposed by Bagnold (1941) based on the assumptions that AA AA is proportional to AA AA 2 * and that the near surface particle velocity increases linearly with AA AA * . This yields an expression for AA AA of the form AA AAAA 3 * + bb . A cubic expression for the integrated mass flux can also be obtained by assuming that particle velocity near the surface is invariant with AA AA * , but considering a linear increase of AA AA with AA AA * (Sørensen, 1991 (Sørensen, , 2004)) . However, experiments show that a cubic increase of AA AA with AA AA * is only likely to happen when saltation develops over rigid beds (Ho et al., 2011) . In Figure 6a , a good agreement is obtained for both polynomial functions, although the quadratic fit is slightly better (root mean square error, RMSE, equal to 0.0036 instead of 0.0043). In fact, for the range of studied friction velocities, small differences between the two functions are obtained. In addition, a good agreement is seen between the integrated mass flux and the product AA AAmmllrr estimated from fitting the particle mass flux profile to an exponential function (Figure 3 ). This agreement underlines the importance of an accurate representation of the near surface mass flux profile. Moreover, it reveals that the contribution of the upper region of the saltation layer to the integrated mass flux is negligible. In Figure 6b , the comparison between simulation results and saltation models is made an impact threshold friction velocity of 0.175 AA m s -1 (the value of AA AA * ,eeee obtained for AA AA * = 0.4 m s -1 , as previously discussed). In the models proposed by Pomeroy and Gray (1990) , Sørensen (2004) and Durán et al. (2011) , the impact threshold friction velocity is a parameter in the integrated mass flux equations which characterizes the erodible bed (Table 4 ). friction velocities lower than AA 0.6 m s -1 , a good agreement is seen between simulation results and the saltation model proposed by Doorschot and Lehning (2002) . At higher friction velocities, the model proposed by Doorschot and Lehning (2002) predicts greater values for AA AA and a better agreement is obtained with the expression proposed by Durán et al. (2011) . AA AADDDDDD scales with AA AA 2 * , which is supported by the current simulation results. However, this equation is highly sensitive to the value of the impact threshold friction velocity and the observed agreement is greatly compromised for different values of AA AA * ,iiii . Bagnold (1941) and Sørensen (2004) proposed expressions for AA AA proportional to AA AA 3 * . When using the coefficients proposed by Vionnet et al. (2014) , a greater mass flux is obtained with Sørensen's expression in comparison with the simulation results. Vionnet et al. (2014) estimated those coefficients from fitting AA AASS∅ to the experimental measurements of Nishimura and Hunt (2000) . Conversely, the expression proposed by Bagnold (1941) to describe saltation over uniform grains ( AA AA = 1.5 ) predicts lower values for AA AA . For friction velocities lower than AA 0.6 m s -1 , the simulation results agree well with the model proposed by Bagnold (1941) Sørensen ( 2004 ) AA AADDDDDD = CC ρρ ff gg DD * ,iiiiDD 2 * ( 1 - DD 2 * ,iiii DD 2 * ) AA AA= 8.5 (b) Durán et al. ( 2011 ) Note. AA AARR is a reference diameter, = 250 × 10 -6 m . (a) Constant parameters proposed by Vionnet et al. (2014) from fitting the equation to the experimental measurements of Nishimura and Hunt (2000) . proposed by Pomeroy and Gray (1990) underestimates the integrated mass flux in comparison with the remaining models and the simulation results. This is partly justified by the authors assumption of a relatively shallow saltation layer (saltation layer height varying from 0.7 to 5 cm for AA AA * varying from 0.3 to AA 0.8 m s -1 ). However, even by adjusting the height of integration from 15 cm to the proposed values, the integrated mass flux obtained with the current numerical model is significantly greater that the evolution proposed by Pomeroy and Gray (1990) . Hence, the deviation between AA AAPP&GG and the remaining models and simulation results is mainly related to the erroneous scaling of the integrated mass flux with AA AA * .
The Effect of Mean Grain Diameter In this section, we continue the analysis of saltation over a bed of equally sized grains. The effect of grain size is studied by comparing the results presented in the previous section (S1, AA ⟨dd⟩ = 200 μμm ) with those from simulations S2, obtained for different grain sizes. The vertical profiles of particle mass flux, concentration and mean streamwise velocity obtained for grain diameters ranging from 200 to 400 AA AAm are presented in Figures 2d-2f . It can be observed that particle streamwise velocity decreases when the grain size increases (Figure 2f ). This is due to the fact that aerodynamic drag applied to the saltating particles increases approximately with AA AA 2 , but particle mass increases with AA AA 3 . Hence, the ability of the flow to accelerate the saltating grains reduces with particle mass. The near surface particle velocity also decreases with the grain diameter (inset in Figure 2f ). Although the near surface particle velocity does not vary significantly with AA AA * , it clearly varies with the grain size. As the grain size increases, the particle mass flux decreases near the surface and increases at higher elevations of the saltation layer (Figure 2d ). Near the surface, this trend is justified by the decrease in particle streamwise velocity as AA ⟨dd⟩ increases (Figure 2f ). Above ∼4 cm, the increase in particle mass flux as the grain size increases is due to the rise in particle concentration (Figure 2e ), which is related to both an increase in particle mass and the number of particles aloft. The vertical profiles of particle mass flux obtained for AA AA * = 0.4 m s -1 are also presented in logarithmic scale in Figure 7a . The results obtained with AA ⟨dd⟩ = 100 μμm are added for comparison. An exponential decay along the saltation layer is clear for the greater grain sizes ( AA ⟨dd⟩ between 200 and 400 AA AAm ), which is in agreement with field measurements (Martin & Kok, 2017) as previously discussed in Section 4.1. The vertical profile obtained with the smallest grain size ( AA ⟨dd⟩ = 100 μμm ) differs significantly from the others. A similar trend inside the saltation layer is visible up to 1 cm height. However, at greater heights, the profile assumes a different shape suggesting transition from saltation to suspension. In fact, for the smallest grain size, particles can be observed up to the top of the domain, while for greater grain sizes, aeolian transport seems to occur via saltation only as the mass flux ceases at approximately 14 cm height. The integrated mass flux is presented in Figure 8a along with the expression proposed by Bagnold (1941) and the numerical results from Doorschot and Lehning (2002) for varying mean grain diameters and friction velocities. Bagnold's expression establishes that AA AA is proportional to AA ⟨dd⟩ 1 2 , following his wind tunnel experiments performed with uniform sand beds characterized by mean diameters ranging from 100 AA AAm to 1 mm. The numerical model of Doorschot and Lehning (2002) also predicts an increase in the integrated mass flux with the grain diameter. In contrast, a negligible variation is obtained with our model for grain diameters ranging from 200 to 400 AA AAm : the reduction in mass flux near the surface and its increase at higher elevations for increasingly bigger grains (Figure 2d ) counterbalance each other. In fact, other saltation models do not predict an explicit variation of AA AA with particle mean diameter (e.g., Durán et al., 2011; Sørensen, 2004) . In opposition to Bagnold's experiments, the wind tunnel measurements carried out by Dong et al. (2003) revealed a reduction in the integrated mass flux with the grain diameter. However, the comparison between sand beds is performed considering the same wind speed at a given reference height. Hence, it is observed that for the same wind speed at the chosen reference height, the integrated mass flux decreases as the grain size increases. In the simulations performed, the imposed friction velocity is kept constant when varying the grain size, which implies different velocities at a given reference height, depending on the mass flux of saltating particles and the respective momentum transfer. The negligible variation of the integrated mass flux with AA ⟨dd⟩ obtained with our model goes along with an increase in the wind speed at all heights as the grain size increases. When analyzing the experiments of Dong et al. (2003) performed with different grain sizes but yielding similar integrated mass fluxes, a greater wind speed is also obtained for greater grain sizes. When considering a uniform bed with grains of 100 AA AAm , a greater integrated mass flux is obtained. However, as previously discussed, particles between 1 and 15 cm height might not be in saltation but rather in suspension. When modeling particles smaller than 200 AA AAm , a rigorous definition of the saltation layer height is needed to fully assess the impact of the mean diameter on the integrated mass flux in saltation. The equilibrium surface friction velocity varies considerably with the mean grain size. In Figure 9a , an increase of AA AA * ,eeee is observed when AA ⟨dd⟩ increases for values greater than 200 AA AAm , which is consistent with the results of Kok and Renno (2009) . For a given AA AA * , the total momentum transfer from the fluid to the particles decreases for greater grain diameters. This is partly due to a smaller number of particles aloft, which overcomes the increase in drag applied on each grain. 4 ) and from the numerical model proposed by Doorschot and Lehning (2002) (D&L) . In (a), the curves are computed considering a uniform bed characterized by different grain diameters. In (b), both a uniform and a mixed-sized bed with a mean grain diameter of AA 200 μμm are considered. MELO ET AL. 10.1029/2021JD035260 19 of 26
The Effect of Mixed-Sized Grains In order to model saltation over a bed of mixed-sized grains, the size distribution of surface grains is described by a lognormal distribution. In this section, the results from simulations S3, obtained with different standard deviations of the grain diameter, are presented and compared with those from simulations S1, obtained with a uniform grain size. The vertical profiles of particle mass flux, concentration and mean streamwise velocity are presented in Figures 2g-2i. Grain size heterogeneity leads to a greater mean particle streamwise velocity, both near the surface and at higher elevations (Figure 2i ). This is due to an increase in the number of smaller particles aloft, which are easily accelerated by the fluid flow. Similarly to Figure 2f (simulations S2), the variation of particle speed close to the surface with AA AA * is negligible; however, a clear variation with the bed characteristics is observed. The effect of the bed size distribution on the particle streamwise velocity profile is less significant when a mass-weighted average is considered. This is due to a reduced contribution of the smaller grains to the average profile. Figures 2g and 2h show that grain size heterogeneity decreases particle mass flux and concentration close to the surface, but leads to greater values at higher elevations of the saltation layer. The vertical profiles of particle mass flux obtained for AA AA * = 0.4 m s -1 are presented logarithmic scale in Figure 7b . As expected, close to the surface, an exponential decay across the saltation layer is observed. At higher elevations, a cloud of suspended grains forms above the saltation layer of mixed-sized beds and a second distinct exponential decay of the mass flux along the height is observed. The transition from saltation to suspension occurs between 5 and 12 cm height, approximately, and is characterized by the change in gradient of the mass flux profiles. This trend was previously observed in field measurements (Gordon et al., 2009) and other numerical models (e.g., Nemoto & Nishimura, 2004) . The probability density function (PDF) of particle size at different heights is presented in Figure 10 for AA AA * = 0.4 m s -1 and both size distributions ( AA AAdd of 100 and 200 AA AAm ). The PDF of particle size at the bed is also presented for comparison (the left tail of the distribution is not obtained, as a minimum grain size of 50 AA AAm is specified in the simulations). Below ∼3 cm height, the size distribution of particles aloft is reasonably well approximated by a lognormal distribution. It is similar to the PDF at the bed, but skewed toward smaller grain sizes. From 5 to 10 cm height, a bi-lognormal distribution is visible in both simulations. In this region, for progressively greater heights, the probability density of smaller grains increases and the probability density of bigger grains decreases. Finally, above ∼14 cm, a new lognormal distribution arises, characterized by grains smaller than 100 AA AAm . The presented variation of particle size distribution with height agrees well with the results of Nemoto and Nishimura (2004) and is related to the saltation-suspension transition observed in Figure 7b . In drifting snow events, particle sublimation can change the size distribution by reducing the size of particles aloft. This modifies the equilibrium saltation state and enhances the transport of particles in suspension. Figure 10b shows that a wider lognormal bed size distribution leads to smaller grain sizes in the first centimeters above the surface. Smaller grains and less particles aloft justify the decrease in mass flux close to the surface AAm present between 8 and 15 cm height is greater. Considering that these particles are transported in saltation, this is in agreement with the increase in saltation layer height observed in Figure 7b . The integrated mass flux is presented in Figure 8b . The simulation results are compared with Bagnold's model, considering different values for the parameter AA AA (Bagnold, 1941) , and with the results of the numerical model proposed by Doorschot and Lehning (2002) . In the latter, particle size is assumed uniform ( AA ⟨dd⟩ = 200 μμm ) or defined by a lognormal distribution ( AA ⟨dd⟩ = 200 μμm , AA AAdd = 200 μμm ). In general, the integrated mass flux obtained with the current model increases with bed heterogeneity. This trend is also predicted by Bagnold (1941) . However, it contrasts with the evolution obtained with the model of Doorschot and Lehning (2002) , in which AA AA decreases when the bed heterogeneity increases. The effect of bed size distribution on the integrated mass flux underlines the importance of correctly describing particle size when estimating snow saltation mass flux. According to the simulation results, this is particularly relevant when AA AA * is greater than AA 0.4 m s -1 . Even though a rigorous of the saltation layer height is not taken in this work, similar trends are obtained when the integration height is limited to the first 10 cm. Moreover, the effect of bed heterogeneity on the computed integrated mass flux is even more significant if the suspension layer is taken into account. The integrated mass flux obtained for a uniform bed of grains with 100 AA AA m in diameter is closer to the values obtained for the studied mixed-sized beds, compared to the other uniform beds with larger grains (Figure 8a ). However, over the uniform bed with grains of 100 AA AAm , particles above 1 cm height seem to be transported in suspension (Figure 7a ). Taking also into account that an increase in the mean particle diameter from 200 to 400 AA AAm leads to a negligible variation of AA AA (Figure 8a ), it is in general not possible to correctly model saltation over a mixed-size bed considering a representative diameter and equally sized grains. An increase in bed heterogeneity also leads to an increase in the equilibrium surface friction velocity, AA AA * ,eeee (Figure 9b ). In contrast with the simulations performed over equally sized grains, AA AA * ,eeee slightly increases with AA AA * . This trend is specially visible for the results obtained with AA AAdd = 200 μμm . For a given AA AA * , the total exchange of momentum from the fluid to the particles decreases for greater standard deviations of the size distribution. Taking into account that the drag applied on each grain is approximately proportional to AA AA 2 and that the number of particles aloft does not vary in a monotonous way with AA AAdd , the decrease in the momentum exchange is explained by the presence of particles with diameters smaller than the mean value ( AA ⟨dd⟩ = 200 μμm ).
The Effect of Interparticle Cohesion We complete the analysis of mixed-sized bed saltation by studying the effect of interparticle cohesion. In this section, the results obtained with simulations S4 are presented. A bed of mixed-sized grains characterized by a lognormal distribution with AA ⟨dd⟩ = 200 μμm and AA AAdd = 100 μμm is considered. The results are compared with those from simulation S3, that were performed with the same particle size distribution but neglecting interparticle cohesion. The vertical profiles of particle mass flux, concentration and mean streamwise velocity are presented in Figures 2j-2l . As cohesion energy increases, particle concentration decreases significantly close to the surface and increases slightly at higher regions of the saltation layer (Figure 2k ). Particle mean streamwise velocity increases with cohesion energy at all heights (Figure 2l ). As expected, close to the surface, a negligible variation of particle streamwise velocity is obtained for different AA AA * ; however, a clear variation with interparticle cohesion is seen. As the ejection velocity increases, the maximum height attained by the saltating particles increases as well. This justifies the observed larger particle concentration at higher elevations. Particle mass flux is given by the product of particle concentration and streamwise velocity. It decreases close to the surface due to a strong reduction in the number of particles and increases at higher regions of the saltation layer due to the rise of both the number of particles aloft and the particle streamwise velocity (Figure 2j ). The equilibrium friction velocity, AA AA * ,eeee , is presented in Figure 9c . It is expected to vary with the bed type, and therefore, with the strength of the interparticle bonds. In fact, AA AA * ,eeee increases with the cohesion energy, which was also obtained by Comola, Gaume, et al. (2019) . Cohesion energy has a direct effect on the number of ejected grains computed from energy conservation, AA AAEE (see Equation 8a ). If AA AAEE becomes smaller than AA AAMM , the number of ejected grains is restricted by energy conservation and it decreases for increasing values of AA AA . Hence, for the same impact velocity and impacting grain diameter, the number of splashed grains reduces with cohesion energy (Comola & Lehning, 2017) . Our results suggest that this leads to a global decrease of particles aloft (Figure 2k ). As a result, for greater values of cohesion energy, the total momentum transfer from the fluid to the particles is smaller (Figure 9c ), as well as the consequent decrease in streamwise wind speed. This leads to a general increase in particle speed (Figure 2l ). The initial velocity at which the splashed grains are ejected from the bed does not vary directly with interparticle cohesion (see distribution characteristics presented in Table 1 ). However, greater impact velocities lead to higher ejection velocities. The integrated mass flux is presented in Figure 11 . The results obtained with the saltation models proposed by Pomeroy and Gray (1990), Doorschot and Lehning (2002) and Sørensen (2004) are also presented for comparison. These models are currently used in atmospheric models, such as RACMO (Lenaerts et al., 2012) , MAR (Amory et al., 2015 (Amory et al., , 2021)) , Alpine3D (Lehning et al., 2008) and Meso-NH (Vionnet et al., 2014) , to estimate snow saltation mass flux. The expressions proposed by Pomeroy and Gray (1990) and Sørensen (2004) are plotted for two limiting values of the impact threshold friction velocity: obtained with simulation S3, AA AAdd = 100 μμm (non-cohesive bed) and with simulation S4, AA AA= 5 × -9 J . As previously explained, in this work, the impact threshold friction velocity is assumed equal to the equilibrium friction velocity at the lowest value of AA AA * that was studied ( AA AA * = 0.4 m s -1 ). The results obtained with the model developed by Doorschot and Lehning (2002) . This is to reduction of particle mass flux close to the surface to its increase at higher elevations as cohesion energy increases (Figure 2j ). At low friction velocities ( AA AA * = 0.4 m s -1 ), the reduction of particle mass flux close to the surface (due to the decrease in particle concentration) prevails, while at greater AA AA * , the rise in mass flux at higher elevations (due to the increase in particle velocity) becomes more significant, leading to a global growth of the integrated mass flux. Naturally, for very high values of cohesion energy, for which particle ejection is highly compromised, the reduction in particle concentration is expected to prevail at all friction velocities, leading to a reduction in the integrated mass flux. A better agreement between the expression proposed by Sørensen (2004) , using the parameters proposed by Vionnet et al. (2014) , and the simulation results is obtained when interparticle cohesion and a lognormal size MELO ET AL. 10.1029/2021JD035260 22 of 26 distribution are considered. This is, when considering a more realistic snow bed. Nonetheless, greater values for AA AA are predicted with AA AASS∅ . An overestimation of the integrated mass flux in saltation is consistent with the overestimation of blowing snow particles obtained by Vionnet et al. (2014 Vionnet et al. ( , 2017)) . The effect of the impact threshold friction velocity on AA AASS∅ is mainly visible at lower friction velocities. At AA AA * = 0.4 m s -1 , the adjustment of the impact threshold friction velocity improves the agreement between model and simulation results obtained with different values for cohesion energy. The results obtained with the numerical model of Doorschot and Lehning (2002) agree well with the simulation results obtained with mixed-sized and cohesionless grains or AA AA= 10 -10 J , over the whole range of the studied friction velocities. Even though a good agreement is also obtained over a bed of uniform grains for AA AA * < 0.6 m s -1 (Figure 6b ), the effect of mean grain diameter and bed heterogeneity on the integrated mass flux predicted by Doorschot and Lehning (2002) is not consistent with the evolution obtained by the present model (Figure 8 ). The expression proposed by Pomeroy and Gray (1990) considerably underestimates the integrated mass flux in comparison with the simulation results and remaining models, independently of the assumed values for the impact threshold friction velocity. The underestimation of the saltation mass flux might be one of the causes for the underestimation of the blowing snow mass flux obtained by et In the drifting snow model proposed by Amory et al. (2021) , the integrated mass flux in saltation is also computed with the expression of Pomeroy and Gray (1990) . However, more reasonable estimates of the blowing snow mass flux are obtained. This is mainly attributed to the improved calculation of the fluid threshold friction velocity and of snow densification induced by the occurrence of drifting snow. Other aspects of the drifting snow model will also influence the particle mass flux in suspension near the surface. They are, for example, the type of lower boundary condition implemented, the turbulence diffusivity considered in the saltation-suspension transition region, and the assumed particle streamwise velocity above the saltation layer.
Conclusions The modeling of snow saltation is particularly challenging due to the metamorphic nature of snow. Depending on the meteorological conditions, snow grains can have multiple shapes and sizes and form interparticle ice bonds between them. During snow transport, the interparticle bonds break and snow particles shape and size change due to fragmentation and sublimation. However, snow saltation models used in mesoscale models generally neglect or oversimplify these particularities, leading to uncertainties in the estimated mass flux that are difficult to quantify. In this work, an LES-based model coupled with state-of-the-art splash functions is used to simulate the complex particle-wind-bed interactions. This approach allows the modeling of steady state saltation over a variety of bed types and the analysis of the effect of grain size and interparticle cohesion on saltation dynamics. The numerical model is able to simulate the main saltation characteristics observed in previous models and experiments: the focus point in the average streamwise wind profiles, an average streamwise particle speed close to the surface invariant with respect to the friction velocity, the exponential decay of particle mass flux with increasing height, and the scaling of the integrated mass flux with the square of the friction velocity. Moreover, as expected, for friction velocities sufficiently greater than the fluid threshold friction velocity, the resulting steady state is characterized by a dynamic equilibrium between splash and deposition. Over mixed-sized beds, different particle size distributions are obtained depending on the distance to the snow surface, as expected when transition from saltation to suspension occurs. The relative importance of snow bed characteristics on saltation dynamics is analyzed by varying the particle size distribution and interparticle bond strength in a systematic way. Bed characteristics, as grain size and interparticle cohesion, significantly influence saltation dynamics, in particular, particle speed, surface friction velocity and integrated mass flux. Particle speed close to the surface is approximately invariant with respect to the friction velocity for all beds that were considered; however, it varies with the bed type. This is relevant for the development of simple saltation models, which are usually based on an assumption for the near surface particle speed. Nevertheless, the mean particle speed in the saltation layer increases with the friction velocity. The average surface friction velocity during steady state saltation, defined here as the equilibrium friction velocity, increases for greater values of the mean grain diameter, standard deviation of the size distribution and interparticle cohesion. The equilibrium friction velocity is tightly correlated with the impact threshold friction velocity, which is an important parameter to estimate saltation mass flux. Over uniform beds, a negligible variation of the integrated mass MELO ET AL.
10.1029/2021JD035260 23 of 26 flux with particle size is obtained for particles ranging between 200 and 400 AA AAm . When considering a mixedsized bed characterized by a lognormal distribution, an increase in the integrated mass flux is seen due to an average increase in particle speed and concentration. The results presented highlight that the integrated mass flux over mixed-sized beds can be hardly reproduced by an equally sized bed with a representative mean diameter -a tempting assumption in simple saltation models. The integrated mass flux also varies with interparticle cohesion, but in a non-monotonous way: it decreases with the strength of interparticle bonds for lower friction velocities and it increases for higher friction velocities. Overall, greater values of cohesion lead to a reduction in the number of particles aloft which, at high wind speeds, is balanced by an increase in particle speed. In general, the greater the friction velocity, the greater the effect of bed properties on saltation characteristics. High wind speed events might be rare in some regions. However, they are responsible for major modifications of the snow cover. The agreement between simulation results and the saltation models typically used in large scale atmospheric models depends on the bed characteristics. For specific bed types, a relatively good agreement with the computed integrated mass flux can be obtained with the models of Sørensen (2004) , using the parameters proposed by Vionnet et al. (2014) , and Doorschot and Lehning (2002) . However, these models either consider fixed parameters, which are not adjustable to the snow type, or predict a different variation of the integrated mass flux with the mean grain size and bed heterogeneity. A systematic underestimation and overestimation of the integrated mass flux is obtained with the expression proposed by Pomeroy and Gray (1990) and Sørensen (2004) , respectively. This might partly justify the underestimation and overestimation of blowing snow mass flux presented, respectively, by Amory et al. (2015) and Vionnet et al. (2014 Vionnet et al. ( , 2017)) . Inaccuracies in the calculation of particle concentration at the top of the saltation layer can also be related to poor estimates of the vertical profile of particle mass flux, the averaged streamwise particle speed and the saltation layer height. Grain size and interparticle cohesion influence all variables of interest. Therefore, improvements on snow transport models can only be reached if all the referred quantities are correctly represented. Further efforts must be made to fully model the effect of bed characteristics on snow saltation. For example, interparticle cohesion is also expected to influence particle ejection velocity during splash and the fluid threshold for the onset of aerodynamic entrainment (Comola et al., 2021) . Moreover, the strength of interparticle bonds between grains that did not leave the surface and between those that failed to rebound might not be the same. From the experimental work side, a correlation between interparticle cohesion and meteorological conditions or measurable snow properties like snow density or snow hardness is still needed. In addition, exhaustive direct comparisons between simulation results and experimental measurements of snow saltation must be performed to complete model validation. In order to better assess the model inner parameters, further studies of the splash process over natural snow beds are required, as well as detailed field measurements characterizing both the wind speed, the snow bed and the particles in saltation. Simple and computationally inexpensive saltation models are much needed in mesoscale models. However, the in depth study of snow saltation is necessary to fully understand the implications of the simplifying assumptions that are used and to estimate the errors they might introduce. This article shows the capabilities of an LES-based model to simulate snow saltation, presents the effect of bed properties on saltation dynamics and motivates further studies in this field. It highlights the limitations of the snow saltation models currently employed in atmospheric models and the need for improved ones that take into account the effect of snow surface characteristics. Without accurate estimations for the mass flux in saltation, atmospheric models will hardly deliver reasonable estimates of blowing snow mass flux and sublimation. Hence, the effect of snow transport and sublimation on large scale mass and energy balances is highly compromised. maximum probability of rebound, equal to 0.9 as proposed by Groot Zwaaftink et al. (2014) for snow particles, [s m -1 ] is a constant set to AA 2 s m -1 (Anderson & Haff, 1991) and | , | [m s -1 ] is the particle velocity at impact. The velocity of rebound, | , | [m s -1
Both the energy and momentum conservation equations are solved for AA AA by statistically representing the kinetic energy and momentum of the ejected grains by their mean values. Only the horizontal direction of
, 5 × 10 -10 , 5 × 10 -9
Figure 1 . 1 Figure1. Vertical profiles of mean streamwise wind speed obtained before saltation onset and during steady state saltation (simulations S1). The inset is a zoom-in to the near surface region during saltation.
Figure 2 . 2 Figure 2. Vertical profiles of particle mass flux, concentration and streamwise velocity obtained with simulations S1 (a-c), S2 (d-f), S3 (g-i) and S4 (j-l). In (d-i) and (j-l) results from simulations S1 and S3 are presented for comparison, respectively. All values are obtained from surface averages and time averages over the last 100 s of each simulation. The insets in (c and f) are a zoom-in to the near surface region.
Figure 3 . 3 Figure 3. Vertical profile of particle mass flux obtained with simulations S1 and fit to exponential decay. The reference height, AA AArr , computed from the exponential fit and from the expression proposed by Nishimura and Hunt (2000) (N&H) is indicated at each exponential curve.
partially related to changes in the snow cover during the experiments. Based on these results, Walter et al. (
Figure 4 . 4 Figure 4. Surface friction velocity obtained with simulations S1. The fluid threshold friction velocity is also presented as a reference. In these simulations, saltation is allowed to develop after the first 25 s. The equilibrium friction velocity is presented in the inset.
Figure 5 . 5 Figure 5. Time-evolution of the mass of particles aloft per unit area (purple line). The time-evolution of the vertical mass flux of particles leaving the surface either through aerodynamic entrainment or splash and the vertical mass flux of particles deposited are presented in blue, dashed black and orange, respectively. Results obtained from simulation S1 with AA AA * = 0.4 m s -1 . The arrows indicate the y-axis corresponding to each curve. The rectangle encloses the time interval used to compute the time-averaged quantities.
to failure of rebound are also presented. The results were obtained for AA AA * = 0.4 m s -1
Figure 6 . 6 Figure 6. Integrated mass flux obtained with simulations S1. The error bar is twice the standard deviation of the results. (a) Fit of simulation results to quadratic and cubic functions (RMSE is the root mean square error of the fit); comparison with the product AA AAmmllrr obtained from the exponential fit of the particle mass flux profile. (b) Comparison with saltation models.
Figure 27 in Durán et al. (2011), assuming a packing fraction of the bed,AAAAbb , equal to 0.95.
Figure 7 . 7 Figure 7. Vertical profiles of particle mass flux obtained with simulations S2 (a) and S3 (b) for AA AA * = 0.4 m s -1 . Results from simulations S1 are presented for comparison.
Figure 8 . 8 Figure 8. Integrated mass flux obtained with simulations S2 (a) and S3 (b) forAA AA * equal to 0.4, 0.6 and 0.8 AA m s -1 . Results from simulations S1 obtained with the same AA AA * are also presented for comparison. To improve readability, some data points are slightly shifted in the AA AA * axis. The error bar is twice the standard deviation of the results. The curves are obtained from Bagnold's model ( AA AABBBBBB in Table 4) and from the numerical model proposed by Doorschot and Lehning (2002) (D&L). In (a), the curves are computed considering a uniform bed characterized by different grain diameters. In (b), both a uniform and a mixed-sized bed with a mean grain diameter of AA 200 μμm are considered.
Figure 9 . 9 Figure 9. Equilibrium friction velocity obtained with simulations S2 (a), S3 (b) and S4 (c). In (a and b), results from simulations S1 are presented for comparison. In (c), results from simulations S3 are presented for comparison.
7b. Moreover, the fraction of grains within the range 200-500 AA
Figure 10 . 10 Figure 10. Probability density function (PDF) of particle size at the bed and at different heights obtained from simulations S3 considering AA AA * = 0.4 m s -1 .
Figure 11 . 11 Figure 11. Integrated mass flux obtained with simulations S4 for AA AA * equal to 0.4, 0.6 and 0.8 AA m s -1 . Results from simulations S3 obtained with the same size distribution and AA AA * are also presented for comparison. To improve readability, some data points are slightly shifted in the AA AA * axis. The error bar is twice the standard deviation of the results. The expressions proposed by Sørensen (2004) and Pomeroy and Gray (1990) (P&G) are plotted for comparison considering different values of the impact threshold friction velocity. The results from Doorschot and Lehning (2002) (D&L) are obtained for a bed characterized by a lognormal distribution with AA ⟨dd⟩ = 200 μμm and AA AAdd = 100 μμm .
Table 1 1 Initial Velocity of Aerodynamically Entrained, Splashed and Rebounding Grains: Distribution Type, Mean and Standard Deviation MELO ET AL. 10.1029/2021JD035260 8 of 26 momentum equation is taken into account as the vertical component of the impact velocity is relatively small
Table 2 2 Parameters of the Splash Model MELO ET AL. 10.1029/2021JD035260 9 of 26
Table 3 3 Simulation Input Parameters MELO ET AL.
if the constant parameter AA AA is increased to 2.8. However, the curve obtained with Integrated mass flux AA [kg m -1 s -1 ] Constant parameters References AA AA AA AABBBBBB = CC AAPP&GG = CC √ ρρ ff gg uu * ,iiiiuu * ⟨dd⟩ dd RR ρρ ff BB uu 3 * ( AASS∅ = ρρ ff gg uu 3 * ( 1 -uu 2 * ,iiii 1 -uu 2 * ) ( uu 2 * ,iiii uu 2 * αα + ββ ) uu 2 * ,iiii uu 2 * + γγ AA uu * uu * ,iiii ) AA AA= 2.8 (highly non-uniform grains) AA= 1.5 (uniform grains) AA AA= 0.68 AA AA= 2.6 , AA AA= 2.5 , AA AA= 2.0 (a) Bagnold (1941) Pomeroy and Gray (1990) AA AA = 2.8 is only expected to describe saltation over a bed of mixed-sized grains. The expression
Table 4 4 Saltation Models for the Integrated Mass Flux, AA AA
|
10.29303/darmadiksani.v2i1.1296
|
cc-by-sa
| null | null |
openalex
|
This catfish-in-the-buckets farming system or "Budikdamber" -stands for "budidaya ikan dalam ember" -is an innovation in home farming that aims to generate the income of village communities affected by the 2018 earthquakes in Selengen Village, North Lombok Regency post Covid-19 pandemic. The Budikdamber fish farming system uses bucket kits to farm freshwater catfish while at the same time grow vegetables as well. The use of cheap bucket kits -as alternative to other conventional farming media -is meant to lower the cost of household's monthly expenses and generate income to support the people's economy in this earthquake-torn village. The Budikdamber fish farming system enables locals to farm not only fish but also intercrops such as spinach and other vegetables right on the lids of the bucket kits used for farming fish inside. The targeted community groups participating in this program are the BLT-DD recipients from Dusun Lembah Berora, Selengen Village, Kayangan District, North Lombok Regency. The products are edible catfish and land spinach that can be harvested for daily consumption or sold to the markets in the hope of generating economic benefits for the locals. The results show that the program has given positive outcomes for the community. Thus, it is hoped that the success of this program will inspire other community groups to implement this Budikdamber fish farming system.Program KKN-PLP ini bertujuan untuk mengintegrasikan ilmu teoritis dan praktis dalam kehidupan nyata yang sesungguhnya dengan maksud agar mahasiswa bertransformasi menjadi pribadi cerdas, mandiri, supel, kaya pengalaman, dan punya ide kreatif dan solutif yang bermanfaat bagi masyarakat di manapun mereka berada. Program budidaya ikan lele dalam ember ini, sebagai salah satu proker dalam KKN-PLP, adalah suatu inovasi untuk meningkatkan pendapatan masyarakat di desa Selengen Lombok Utara pasca pandemi Covid-19. Budidaya ikan lele ini dilakukan menggunakan media ember yang bertujuan untuk meminimalkan biaya pengeluaran rumah tangga namun dapat mendatangkan penghasilan bagi masyarakat minimal untuk kebutuhan sehari-hari. Tidak hanya itu, budidaya ikan lele dalam ember ini juga bisa dimanfaatkan untuk tumpangsari yakni menanam tanaman sela berupa sayuran kangkung darat di atas tutup ember. Masyarakat pengelola program Budikdamber ini yaitu kelompok masyarakat penerima BLT-DD di dusun Lembah Berora, desa Selengen, kecamatan Kayangan kabupaten Lombok Utara. Kelompok masyarakat ini dipilih agar memudahkan pengontrolan dalam pembudidayaan Budikdamber. Produk yang dihasilkan dari program budidaya ini adalah ikan lele dan kangkung darat yang dapat dikonsumsi sehari-hari dan dijual ke pasar untuk meningkatkan pendapatan masyarakat setempat. Hasil evaluasi program menunjukkan bahwa kegiatan ini telah mencapai target yang diharapkan. Setelah mengikuti program ini, kelompok masyarakat yang sudah berhasil membudidayakan Budikdamber diharapkan dapat menginspirasi dan mengedukasi kelompok masyarakat lainnya untuk dapat menerapkan sistem Budikdamber ini.
PENDAHULUAN Mahasiswa fakultas keguruan dan ilmu pendidikan tidak hanya perlu dibekali dengan kemampuan softskill namun juga kemampuan hardskill berupa kemampuan untuk dapat menghadapi tantangan di dunia kerja dan di masyarakat. Galbreath (1999) mengatakan bahwa kita sedang menghadapi sebuah era penuh dengan ketidakpastian di abad 21 ini. Oleh sebab itu, maka (institusi) pendidikan tinggi perlu menyiapkan kecakapan masa depan yang dapat berguna dalam kehidupan peserta didik (Duderstadt, 1999) . Velez (2012) pun mengatakan bahwa ekonomi global saat ini menuntut masyarakat berpendidikan tinggi yang tidak hanya mampu menguasai softskill tapi juga hardskill yang bermanfaat bagi mereka di dunia kerja. Perdebatan antara para pelaku bisnis dan institusi pendidikan terkait mana yang lebih prioritas masih ada, namun dikotomi antara keduanya tidak seharusnya terjadi sebagaimana dinyatakan oleh Alshare dan Sewailem (2018) . Kurikulum Merdeka Belajar Kampus Merdeka (MBKM) memiliki semangat yang sama dengan pernyataan di atas yakni untuk Vol. 2, No.1, Juni 2022, Hal. 24-34. menciptakan lulusan yang cakap dalam kedua ketrampilan tersebut. Persepsi mahasiswa terhadap program MBKM jelas menunjukkan adanya optimisme dan animo yang besar dari mahasiswa terhadap pengimplementasian program ini (Putera & Sugianto, 2020) . Tak dapat dipungkiri bahwa pandemi Covid-19 berdampak negatif terhadap kondisi ekonomi khususnya bagi masyarakat pedesaan. Oleh sebab itu, penguasaan keterampilan kerja untuk menunjang karir dan kehidupan menjadi sangat penting bagi masyarakat banyak untuk dapat bertahan menghadapi gelombang ekonomi yang tidak menentu terutama di abad 21 dan pasca pandemi Covid-19 ini. Program KKN-PLP ini bertujuan mengintegrasikan penerapan ilmu teoritis dan ilmu praktis dalam dunia nyata agar mahasiswa bisa bertransformasi menjadi pribadi cerdas, mandiri, supel, kaya pengalaman, dan dapat memberi solusi bagi masyarakat di manapun mereka berada. Kegiatan ini pun sangat sesuai dengan harapan dan tujuan pembangunan berkelanjutan yang dicanangkan PPB dalam 17 tujuan pokok SDGs (Sustainable Development Goals) di mana di dalamnya terdapat tujuan ingin "Menghapus kemiskinan" (No poverty, No. 1), "Menghapus kelaparan" (No hunger, No. 2), dan "Menciptakan pertumbuhan ekonomi dan pekerjaan yang baik" (Decent work and economic growth, No. 8) (UN Department of Economic and Social Affairs, May 2015, https://sdgs.un.org/goals). Relevan dengan paparan di atas, maka salah satu proker (program kerja) yang diajukan oleh tim KKN-PLP mahasiswa Pendidikan Keguruan dan Ilmu Pendidikan ini selain mengajar anakanak desa adalah berupa edukasi pengenalan dan penerapan sistem budidaya ikan lele dalam ember atau diakronimkan dengan nama "Budikdamber" yang bisa ditumpangsarikan dengan tanaman sayuran lain seperti kangkung darat dan sejenisnya. Program budidaya ikan lele dalam ember ini adalah suatu inovasi yang terbukti dalam beberapa penelitian dan pengabdian masyarakat dapat: (1) meningkatkan pendapatan masyarakat desa, (2) membantu mengurangi dampak akibat kesulitan ekonomi, dan (3) bisa menjadi peluang bisnis yang menjanjikan jika diternak dan dikelola secara berkelanjutan (Ulya, 2021; Saputri & Rachmawatie, 2020) terutama di daerah yang terdampak gempa seperti desa Selengen Lombok Utara. Sistem Budikdamber ini dilakukan menggunakan media ember yang dimaksudkan untuk meminimalkan biaya pengeluaran rumah tangga dengan proses dan bahan-bahan berbiaya murah pada lahan sempit, namun dapat mendatangkan penghasilan bagi masyarakat minimal untuk memenuhi kebutuhan sehari-hari seperti untuk laukpauk. Tidak hanya itu, pembudidayaan ikan lele dalam ember ini juga Vol. 2, No.1, Juni 2022, Hal. 24-34. bisa dimanfaatkan untuk tumpangsari yakni menanam tanaman sela berupa sayuran kangkung darat di atas tutup ember. Budikdamber cocok untuk dimanfaatkan pada lahan sempit (Nebore, 2021) . Hal ini dapat dilakukan oleh semua orang dan di mana saja. Selain itu, Budikdamber adalah satu upaya alternatif yang mampu membantu meningkatkan pendapatan masayarakat desa dengan resiko kegagalan yang rendah serta mudah diterapkan sebagai usaha sampingan masyarakat. Budikdamber memadukan antara budidaya ikan dan tanaman sayur dalam satu ember (Setiyaningsih, 2020; Kuncoro, 2021; Scabra, Wahyudi, & Rozi, 2021) . Ember digunakan sebagai wadah budidaya degan menerapkan sistem akuaponik dan juga dapat memanfaatkan air sebagai media untuk pertumbuhan tanaman sayuran. Dalam sistem Budikdamber, tidak semua jenis ikan dapat dibudidayakan dengan menggunakan teknik ini. Oleh sebab itu, jenis ikan yang dipilih oleh tim program yaitu jenis ikan lele sebab ikan lele merupakan salah satu jenis ikan yang tahan akan oksigen rendah (Saputri & Rachmawatie, 2020) . Beberapa manfaat ikan lele yaitu dapat menurunkan berat badan, menjaga kesehatan otak, mencegah anemia, menjaga kesehatan jantung (Alodokter.com, Juni 2021, https://www.alodokter.com). Sementara untuk tanaman sela dipilih sayur kangkung darat yang mudah tumbuh di air, bergizi, dan makanan favorit hampir semua masyarakat Lombok. Di antara manfaat kangkung adalah mencegah anemia, meningkatkan imun, melawan radikal bebas, menurunkan tekanan darah, kesehatan mata, dan mengatur keseimbangan cairan (Alodokter.com, Januari 2021, https://www.alodokter.com).
METODE KEGIATAN Pelaksanaan kegiatan pengabdian masyarakat melalui Kuliah Kerja Nyata (KKN) PLP FKIP Universitas Mataram dilakukan dengan metode sosialiasi dan praktek secara langsung pemeliharaan ikan lele dengan sistem Budikdamber dan menanam tanaman sela berupa sayuran kangkung darat pada tutup ember.
Waktu dan Tempat Kegiatan Budikdamber dalam program KKN-PLP ini dilaksanakan dari bulan November 2021-Januari 2022 mulai dari penyerahan dan survei lokasi sampai diakhiri dengan panen lele dan kangkung pada acara penarikan. Program ini dilaksanakan di RT 01, dusun Lembah Berora, desa Selengen, kecamatan Kayangan, kabupaten Lombok Utara. Jarak lokasi dari kampus Universitas Mataram kurang lebih 69KM dengan jarak tempuh menggunakan mobil 1 jam 44 menit. Posko KKN-PLP berada di dalam kompleks kantor Kepala Desa Selengen. Vol. 2, No.1, Juni 2022, Hal. 24-34. Gambar 1. Google maps lokasi KKN-PLP desa Selengen Lombok Utara. (Sumber: https://www.google.com) Desa selengen merupakan salah satu desa di kecamatan Kayangan kabupaten Lombok Utara, berlokasi di ujung timur kecamatan Kayangan dengan wilayah 2240 KM 2 . Desa Selengen memiliki 13 dusun yaitu, dusun Panggung Barat, dusun Panggung Timur, dusun Lembah Berora, dusun Tampes, dusun Selengen, dusun Gubuk Baru, dusun Sambik Jengkel Barat, dusun Sambik Jengkel Perigi, dusun Dompo Indah, dusun Sambik Jengkel Timur, dusun Lokok Mandi, dusun Sangiang, dan dusun Tangga (http://desaselengen.blogspot.com). Untuk lokasi kegiatan budidaya Budikdamber ini diadakan di dusun Lembah Berora dengan memilih 9 KK penerima BLT-DD sebagai Hal ini bertujuan agar tim dan peternak lebih mudah dan dalam mengontrolnya.
Tahap Sosialisasi Sebelum memulai program utama KKN-PLP di desa Selengan kami melakukan survei ke lokasi yang bertempat di dusun Lembah Berora. Setelah tahap survei selesai, tim melakukan sosialisasi yang bertujuan untuk mengedukasi masyarakat dan membimbing mereka tentang sistem Budikdamber. Sosialisasi dan program utama kami dari KKN-PLP desa Selengen hanya memfokuskan bagi masyarakat yang menerima BLT-DD di desa Lembah Berora yakni sebanyak 8 orang. Sedangkan di Karang Bajo tahap sosialisasi dilakukan ke semua dusun dan program utamanya hanya difokuskan di satu dusun saja di rumah Bapak RT 01 dusun Lembah Berora. Gambar 2. Sosialisasi Budidaya Ikan Lele Dalam Ember (Budikdamber). Vol. 2, No.1, Juni 2022, Hal. 24-34.
Praktek Lapangan Pada kegiatan program Budikdamber, ada beberapa tahapan yang dilakukan yaitu:
a. Tahap Persiapan Alat dan Bahan Pada tahap ini dipersiapkan alat dan bahan yang digunakan dalam Budikdamber. Adapun alat dan bahan yang digunakan adalah gelas plastik, gunting, solder, pisau, air, cocopeat, benih ikan lele, pakan lele, dan bibit kangkung darat, dan ember plastik.
b. Tahap Persiapan Benih Ikan Lele dan Bibit Kangkung Darat Benih ikan lele yang dipilih merupakan benih ikan lele yang di beli dengan harga Rp. 400/ekor. Benih ikan lele mudah ditemukan dan harganya relative murah dan terjangkau. Ikan lele merupakan ikan yang tahan dengan kadar oksigen yang sedikit sehingga cocok digunakan untuk Budikdamber. Bibit kangkung yang digunakan dalam Budidaya ini merupakan bibit kangkung yang di beli. Tanaman kangkung dipilih untuk budidaya ini, dikarenakan mudah dibudidayakan dengan hidroponik.
c. Tahap Pembuatan Wadah Budikdamber Adapun langkah-langkah budidaya ikan lele dalam ember adalah sebagai berikut: 1. Menyiapkan alat danbahan. 2. Melubangi gelas plastik sebanyak 8 buah dengan solder pada bagian bawah dan samping gelasplastik. 3. Memasukkan kangkung dengan akarnya sebesar kurang lebih 10 cm dan diisikan cocopeat. 4. Melubangi tutup ember. 5. Mengisi ember dengan air diamkan selama satu hari. 6. Memasukkan bibit ikan lele ke dalam ember diamkan sampai satu hari. 7. Merangkai gelas kangkung dan dimasukkan ke dalam lubang yang sudah dilubangi di bagian tutupember. 8. Mengulangi langkah 1-7 dengan menggunakan ember yang lain untuk budidaya ikan lele dalam ember.
d. Tahap Monitoring dan Pengontrolan Budidaya Ikan Lele Monitoring dan pengontrolan bertujuan untuk melihat perkembangan ikan dan tanaman sayuran kangkung. Monitoring dan pengontrolan ini dilakukan 3 kali dalam seminggu. Vol. 2, No.1, Juni 2022, Hal. 24-34. e. Tahap Pemanenan Ikan lele dapat dipanen minimal berusia 2 Bulan. Sedangkan sayuran kangkung dapat dipanen ketika usia 2 minggu atau 14 hari setelah ditanam.
HASIL KEGIATAN DAN PEMBAHASAN Kegiatan awal dalam program ini yaitu meminta izin ke kantor kepala desa Selengen, kecamatan Kayangan, kabupaten Lombok Utara. Adapun tanggapan dari staf desa dan warga desa mengenai Budikdamber ini sangat mengapresiasi mahasiswa dan mendukung karena sistem ini memiliki tingkat kerugian yang rendah, bisa untuk konsumsi sehari-hari, bisa dipasarkan agar pendapatan masyarakat bertambah karena seperti diketahui harga ikan lele cukup tinggi. Budikdamber merupakan teknologi produksi ikan yang dilakukan pada lahan yang sempit. Teknologi ini dapat dikembangkan bersama dengan teknologi aquaponik sehingga dapat mejadi sumber pemasok kebutuhan protein hewani dan nabati dalam satu siklus produksi yang sama. Mengintegrasikan budidaya ikan dan sayuran sekaligus pada yang terbatas lebih menguntungkan dibandingkan dengan teknik budidaya konvensional. Hal tersebut karena dalam satu kali siklus produksi, dapat dihasilkan ikan dan tumbuh-tumbuhan sekaligus. Budidaya sistem akuaponik pada prinsipnya menghemat penggunaan lahan dan meningkatkan efisiensi pemanfaatan hara dari sisa pakan dan metabolisme ikan. Sistem ini merupakan sistem budidaya ikan yang ramah lingkungan 4. Memindahkan bibit ikan lele ke dalam ember yang sudah diisi air. 5. Menanam bibit sayuran kangkung darat ke dalam lubang tutup ember. Gambar 4. Bibit sayur kangkung darat.
Penebaran Benih Ikan Lele dan Kangkung Darat Kegiatan yang dilakukan selanjutnya yaitu persiapan media budikdamber. Ember diisi dengan air kurang lebih 60 liter. Gelas plastik diisi dengan cocopeat sebagai media untuk menanam sayur kangkung darat. Setiap ember diisi dengan sekitar 12 bibit ikan lele dan 8 buah kangkung darat.
Monitoring dan Pengontrolan Budidaya Ikan Lele Monitoring dan pengontrolan bertujuan untuk melihat perkembangan ikan dan tanaman sayuran kangkung. Monitoring dan pengontrolan ini dilakukan 3 kali dalam seminggu.
Panen Ikan Lele dan Kangkung Darat Sayuran kangkung darat pertama kali dapat dipanen ketika usia 2 minggu atau 14 hari setelah di tanam. Hasil awal panen sayur kangkung darat kurang lebih satu ikat per ember. Sedangkan ikan lele dapat dipanen minimal setelah berusia 2 bulan seperti tampak pada gambar 6. Gambar 6. Hasil panen ikan lele dengan sistem budidaya Budikdamber.
Monitoring dan Evaluasi oleh DPL Tim pengabdian dalam program KKN-PLP Desapreneur di desa Selengen ini dibimbing oleh seorang DPL (Dosen Pembimbing Lapangan) yakni Bapak Lalu Jaswadi Putera, M.Pd. DPL membimbing tim mulai dari tahap penyusunan proposal, konsultasi, pelaksanaan kegiatan, pelaporan, sampai tahap evaluasi. DPL melakukan monitoring dan evaluasi selama tiga kali dalam satu periode dengan mendatangi lokasi posko KKN-PLP secara onsite.
KESIMPULAN DAN SARAN Pengabdian masyarakat dengan program utama pengajaran (PLP) dan budidaya ikan lele dalam ember (KKN) dapat dikatakan telah mencapai keberhasilan. Hal ini dapat dilihat dari beberapa indikator yakni: (1) menambah pengetahuan masyarakat terdampak gempa di Vol. 2, No.1, Juni 2022, Hal. 24-34. desa Selengen Lombok Utara tentang sistem budidaya ikan lele dalam ember dengan tanaman sela kangkung; dan (2) meningkatkan pendapatan masyarakat dan meningkatkan kemandirian pangan keluarga dengan memanfaatkan pekarangan rumah menggunakan sistem hidroponik. Terakhir, tim pengabdian program KKN-PLP Desapreneur menyarankan agar sistem budidaya ikan lele dalam ember (Budikdamber) ini dapat diterapkan secara berkelanjutan dan lebih luas jangkauannya dan kelompok masyarakat yang sudah sukses dengan budidaya ini dapat menginspirasi dan mengedukasi komunitaskomunitas di desa-desa lainnya agar bisa meraih kesuksesan yang sama.
UCAPAN TERIMAKASIH Ucapan terimakasih yang sebesarnya-besarnya kami ucapkan kepada pihak Universitas Mataram dalam hal ini Bapak Rektor Universitas Mataram, Bapak Dekan FKIP, beserta pihak LPPM yang telah menyelenggarakan kegiatan KKN-PLP ini dengan baik. Tak lupa pula kami ucapkan terimakasih kepada semua pihak desa Selengen Kec. Kayangan Kab. Lombok Utara yang telah menerima kami dengan baik sejak penyerahan sampai penarikan. Semoga kebaikan Bapak dan Ibu semua diberi ganjaran pahala yang berlimpah di sisi Allah SWT. Aamiin. Gambar 7 . 7 Gambar 7. Monev oleh DPL di kantor desa Selengen Kabupaten Lombok Utara.
Vol. 2, No.1, Juni 2022, Hal. 24-34. Gambar 3. Proses melubangi gelas plastik. 1. Pembuatan Wadah Budidaya Ikan Lele Dalam Ember Wadah Budikdamber dibuat menggunakan ember berukuran sekitar 70 liter. Pembuatan wadahnya mengikuti langkah-langkah berikut: 1. Menutup ember dilubangi menjadi 8 lubang dan satu lubang besar di tengah tutup ember. 2. Melubangi gelas plastik sebanyak 8 buah menggunakan solder pada bagian bawah dan samping gelas plastik agar tanaman lebih mudah memanfaatkan nutrisi dari kotoran ikan. 3. Memasukkan cocopeat ke dalam gelas plastik sebagai media tanam bersama dengan bibit sayuran kangkung darat ke dalam gelas plastik yang sudah diisi dengan cocopeat.
Vol. 2, No.1, Juni 2022, Hal. 24-34.
|
10.5281/zenodo.3336364
|
cc-by
| null | null |
openalex
|
In 200,000 years old human history of existence on planet earth humans have evolved and have been striving to get better every day. In this process humans have altered every natural method to match there purposes and satisfy their needs. Humans therefore have reworked every irrigation process, cultivation process, river systems ETC; and have moved towards much more productivity methods in every field of life but humans couldn't alter the weather according to their desire. Weather has been an important factor for any process to occur on this magnificent and beautiful planet. The word weather roughly means -the state of the atmosphere at a particular place and time as regards heat, cloudiness, dryness, sunshine, wind, rain ETC‖. Of late humans have tried to alter the weather according to their desire to fulfil their needs since late 1990. In our project we are trying to perceive the relation between -weather, irrigation and rainfall‖. This topic is a part of climate engineering, rainfall engineering and Irrigation engineering which are new streams of civil engineering domain. This civil engineering project gives an overview of the modern methods which have been recently adopted by various developed countries to make weather or precipitation act as they desire for. Hence, it means they have hacked the natural process of precipitation and made it artificial. Our project also tries to know the plight of farmers facing drought and floods due to irregularities in rainfall and steps taken by various central and state government organisations to rescue them from this crisis .But to our fate our government is least bothered about the farmers and hence leading to huge loss of population of this beautiful nation and its honest citizens by disasters which leave them helpless and hence in many cases farmers take extreme steps such as suicide and moving towards urbanisation. Our project also consists of photographic references and various other data collected from meteorological department, forest department, irrigation department, land records department, Agriculture department and RTI.irrigated or the place where precipitation is needed. Our states Andhra Pradesh and Telangana have been 2 nd in statics of 2012 suicide estimations .In recent two years 2015 and 2016 the number has gone very high and we are next to only Maharashtra which also shares our fate of unseasonal rains and drought .This problems can be solved by these methods. And further restore the faith of farmers in irrigation and our government also. We as civil engineers must feel responsible and try to implement these path braking and out of the box methods so that these we can be out of this crisis by some extent till an permanent solution of green future is made possible. Cloud Type LWC(g/m The objective of our project is to better understand how irrigation can effect rainfall and also how we can plan an agriculture to feed our growing population .It will also allow us to -Model, Predict and ADAPT‖ to changing climate. It also focuses on how artificial and natural methods of precipitation can make agriculture possible in drought hit zones. Precipitation: The objective of our project is mainly based on studying rainfall (or) in technical terminology it can be described as -Precipitation‖. The history of precipitation goes back to 4.5 billion years ago when our planet broke from sun to form a land mass which was very hot back then but gradually cooled down which resulted in formation of gaseous clouds which ultimately lead to rains. Clouds: In meteorology, a cloud is an aerosol comprising a visible mass of minute liquid droplets or frozen crystals, both of which are made of water or various chemicals. The droplets or pa1rticles are suspended in the atmosphere above the surface of a planetary body. On Earth, clouds are formed by the saturation of air in the hemispheres (which includes the troposphere, stratosphere, and mesosphere). The air may be cooled to its dew point by a variety of atmospheric processes or it may gain moisture (usually in the form of water vapour) from an adjacent source. Nephology is the science of clouds which is undertaken in the cloud physics branch of meteorology. How do we calculate the Amount of Water Present in Cloud: The liquid water content (LWC) is the measure of the mass of the water in a cloud in a specified amount of dry air. It is typically measured per volume of air (g/m3) or mass of air (g/kg). (Bohren, 1998) . This variable is important in figuring out which types of clouds are likely to form and is strongly linked to three other cloud microphysical variables: the cloud drop effective radius, the cloud drop number concentration, and the cloud drop size distribution. (Wallace, 2006) . Being able to determine the cloud formations that are likely to occur is extremely useful for weather forecasting as cumulonimbus clouds are related to thunderstorms and heavy rain whereas cirrus clouds are not directly associated with precipitation Problems with Present Day Precipitation Patterns: As temperatures rise and the air becomes warmer, more moisture evaporates from land and water into the atmosphere. More moisture in the air generally means we can expect more rain and snow (called precipitation) and more heavy downpours. But this extra precipitation is not spread evenly around the globe, and some places might actually get less precipitation than they used to get. That's because climate change and global warming. On average, the world is already getting more precipitation now than it did 100 years ago: 6 per cent more in the United States and nearly 2 per cent more worldwide. This has a huge effect on farmers as many of agricultural schemes in our country and south Asia depend on monsoons and seasonal rains but due to global warming there has been huge change in patterns of rains since last 10 to 20 years.
Scope of the Study: Some Basic The most common chemicals used for cloud seeding include silver iodide, potassium iodide and dry ice (solid carbon dioxide). Liquid propane, which expands into a gas, has also been used. This can produce ice crystals at higher temperatures than silver iodide. After promising research, the use of hygroscopic materials, such as table salt, is becoming more popular. When Cloud seeding increases snowfall takes place when temperatures within the clouds are between 19 and -4 F (-7 and -20 C).Introduction of a substance such as silver iodide, which has a crystalline structure similar to that of ice, will induce freezing nucleation. In midlatitude clouds, the usual seeding strategy has been based on the fact that the equilibrium vapour pressure is lower over ice than over water. The formation of ice particles in super cooled clouds allows those particles to grow at the expense of liquid droplets. If sufficient growth takes place, the particles become heavy enough to fall as precipitation from clouds that otherwise would produce no precipitation. This process is known as "static" seeding. Seeding of warm-season or tropical cumulonimbus (convective) clouds seeks to exploit the latent heat released by freezing. This strategy of "dynamic" seeding assumes that the additional latent heat adds buoyancy, strengthens updrafts, ensures more low-level convergence, and ultimately causes rapid growth of properly selected clouds. Cloud seeding chemicals may be dispersed by aircraft or by dispersion devices located on the ground (generators or canisters fired from anti-aircraft guns or rockets). For release by aircraft, silver iodide flares are ignited and dispersed as an aircraft flies through the inflow of a cloud. When released by devices on the ground, the fine particles are carried downwind and upward by air currents after release. An electronic mechanism was tested in 2010, when infrared laser pulses were directed to the air above Berlin by researchers from the University of Geneva. The experimenters posited that the pulses would encourage atmospheric sulphur dioxide and nitrogen dioxide to form particles that would then act as seeds.
Effectiveness: New technology and research has produced reliable results that make cloud seeding a dependable and affordable water-supply practice for many regions. While practiced widely around the world, the effectiveness of cloud seeding is still a matter of academic debate. In 2003 the US National Research Council (NRC) released a report stating, "...science is unable to say with assurance which, if any, seeding techniques produce positive effects. In the 55 years following the first cloud-seeding demonstrations, substantial progress has been made in understanding the natural processes that account for our daily weather. Yet scientifically acceptable proof for significant seeding effects has not been achieved. Referring to the years 1903, 1915, 1919, 1944, and 1947 weather modification experiments, the Australian Federation of Meteorology discounted "rain making". By the 1950s, the CSIRO Division of Radio physics switched to investigating the physics of clouds and had hoped by 1957 to better understand these processes. By the 1960s, the dreams of weather making had faded only to be re-ignited post-corporatisation of the Snowy Mountains Scheme in order to achieve "above target" water. This would provide enhanced energy generation and profits to the public agencies that are the principal owners. Cloud seeding has been shown to be effective in altering cloud structure and size and in converting super cooled liquid water to ice particles. The amount of precipitation due to seeding is difficult to quantify. There is statistical evidence for seasonal precipitation increases of about 10 per cent with winter seeding. Clouds were seeded during the 2008 Summer Olympics in Beijing using rockets, to coax rain showers out of clouds before they reached the Olympic city so that there would be no rain during the opening and closing ceremonies, although others dispute their claims of success. A 2010 Tel Aviv University study claimed that the common practice of cloud seeding to improve or induce rainfall, with materials such as silver iodide and frozen carbon dioxide, seems to have little if any impact on the amount of precipitation. A 2011 study suggested that airplanes may produce ice particles by freezing cloud droplets that cool as they flow around the tips of propellers, over wings or over jet aircraft, and thereby unintentionally seed clouds. This could have potentially serious consequences for particular hail stone formation.
Impact on Environment and Health: With an NFPA 704 health hazard rating of 2, silver iodide can cause temporary incapacitation or possible residual injury to humans and mammals with intense or continued but not chronic exposure. However, there have been several detailed ecological studies that showed negligible environmental and health impacts. The toxicity of silver and silver compounds (from silver iodide) was shown to be of low order in some studies. These findings likely result from the minute amounts of silver generated by cloud seeding, which are about one percent of industry emissions into the atmosphere in many parts of the world, or individual exposure from tooth fillings. Accumulations in the soil, vegetation, and surface runoff have not been large enough to measure above natural background .A 1995 environmental assessment in the Sierra Nevada of California and a 2004 independent panel of experts in Australia confirmed these earlier findings. "In 1978, an estimated 2,740 tonnes of silver were released into the US environment. This led the US Health Services and EPA to conduct studies regarding the potential for environmental and human health hazards related to silver. These agencies and other state agencies applied the Clean Water Act of 1977 and 1987 to establish regulations on this type of pollution."
International Journal of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 4 Cloud seeding over Kosciuszko National Park a biosphere reserve is problematic in that several rapid changes of environmental legislation were made to enable the trial. Environmentalists are concerned about the uptake of elemental silver in a highly sensitive environment affecting the pygmy possum among other species as well as recent high level algal blooms in once pristine glacial lakes. Research 50 years ago and analysis by the former Snowy Mountains Authority led to the cessation of the cloud seeding program in the 1950s with nondefinitive results. Formerly, cloud seeding was rejected in Australia on environmental grounds because of concerns about the protected species, the pygmy possum. Since silver iodide and not elemental silver is the cloud seeding material, the claims of negative environmental impact are disputed by peer-reviewed research as summarized by the international Weather Modification Association.
Ice Breaking Boom: In many cold countries there is scarcity of fresh water and in many cases due to hail storms and snow, crops get destroyed hence farmers incur huge losses so these kind of methods help in converting hail storms into rain which leads to fresh water irrigation in places like these. In this process and cannon is placed at a high altitude so that it is audible at every place another reason why it is kept at higher altitude is that it can be nearer to clouds as its main effect is on clouds and rains. In many places clouds carry hail storms which eventually effects cultivation of fruits which are main crops to be sown in these places. An cannon is placed at high altitude which is been filled with gun powder at lower part of cannon .This gun powder is burnt which leads to an huge boom sound all over the place this shock wave disturbs the snow present in cloud and hence initiates rainfall .
Effectiveness: This is very effective in places where hails storms are common as these boom sounds can disintegrate huge masses of snow very effectively Hence they can cause rains immediately but these cannot be used in dry and warm places as this equipment is not effective there.
Uses Worldwide: At present France is the only main user of this particular technique but in some parts of United States of America this technique has been gaining popularity as this doesn't have any side effects on nature.
Rain Rockets: This method is similar to that of cloud seeding by aircraft but in this instead of aircrafts rockets are used as these doesn't require much of economical invest and is easily affordable. Many countries have been using this method as these rockets have wide reach with minimal effect and these also can be transported easily without any hassle.
Floods and Drought: Today, droughts and floods are a common feature and their co-existence poses a potent threat, which cannot be eradicated but has to be managed. Transfer of the surplus monsoon water to areas of water deficit is a potential possibility. This would also help create additional irrigational potential, the generation of hydropower, as well as overcoming regional imbalances. The recurrence of drought and famines during the second half of the 19th century necessitated the development of irrigation to give protection against the failure of crops and to reduce large-scale expenditure on famine relief.
Floods in India: Floods are recurrent phenomena in India. Due to different climatic and rainfall patterns in different regions, it has been the experience that, while some parts are suffering devastating floods, another part is suffering drought at the same time. With the increase in population and development activity, there has been a tendency to occupy the floodplains, which has resulted in damage of a more serious nature over the years. Often,
International Journal of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 5 because of the varying rainfall distribution, areas which are not traditionally prone to floods also experience severe inundation. Thus, floods are the single most frequent disaster faced by the country. Flooding is caused by the inadequate capacity within the banks of the rivers to contain the high flows brought down from the upper catchments due to heavy rainfall. Flooding is accentuated by erosion and silting of the river beds, resulting in a reduction of the carrying capacity of river channels; earthquakes and landslides leading to changes in river courses and obstructions to flow; synchronization of floods in the main and tributary rivers; retardation due to tidal effects; encroachment of floodplains; and haphazard and unplanned growth of urban areas. Some parts of the country, mainly coastal areas of Andhra Pradesh, Orissa, Tamil Nadu and West Bengal, experience cyclones, which are often accompanied by heavy rainfall leading to flooding. Area Prone to Flood: In 1980, Rashtriya Barh Ayog (National Commission on Floods) assessed the total area liable to flooding in the country as 40 million hectares (ha), which constitutes one-eighth of the country's total geographical area. The Working Group on Flood Control Programme set up by the Planning Commission for the Tenth Five Year Plan put this figure at 45.64 million ha. About 80 per cent of this area, i.e. 32 million ha, could be provided with a reasonable degree of protection.
Damage from Floods: More significant than the loss of life and damage to property is the sense of insecurity and fear in the minds of people living in the floodplains. The after-effects of flood, such as the suffering of survivors, spread of disease, non-availability of essential commodities and medicines and loss of dwellings, make floods the most feared of the natural disasters faced by humankind. 1955, 1971, 1973, 1977, 1978, 1980, 1984, 1988, 1989, 1998, 2001 and 2004 . Highlights of the damage are given below: Flood-prone areas are shown in the map.
Institutional Arrangements: At the central level, the Union Ministry of Water Resources is responsible for development, conservation and management of water as a national resource, i.e. for general policy on water resources development and for technical assistance to the states on irrigation, multipurpose projects, groundwater exploration and exploitation, command area development, drainage, flood control, water-logging, sea-erosion problems, dam safety and hydraulic structures for navigation and hydropower. It also oversees the regulation and development of inter-state rivers. These functions are carried out through various central organizations. Urban water supply and sewage disposal is handled by the Ministry of Urban Development, while rural water supply comes under the purview of the Department of Drinking Water under the Ministry of Rural Development. The subject of hydro-electric and thermal power is the responsibility of the Ministry of Power. Pollution and environment control comes under the purview of the Ministry of Environment and Forests. Water being a state subject, the state government has primary responsibility for use and control of this resource. The administrative control and responsibility for development of water rest with the various state departments and corporations.
National Water Policy: The National Water Policy adopted by the National Water Resources Council in April 2002 highlights the provisions for project planning, surface-and groundwater development, irrigation and flood control. Irrigation plays a major role in increasing the production of food grains. The policy provides following directives for irrigation management: Irrigation planning either in an individual project or in a basin as a whole should take into account the irrigability of land, cost-effective irrigation options possible from all available sources of water and appropriate irrigation techniques for optimizing water-use efficiency. Irrigation intensity should be such as to extend the benefits of irrigation to as large a number of farming families as possible, keeping in view the need to maximize production; There should be close integration of water-and land-use policies. Water allocation in an irrigation system should be done with due regard to social equity and justice. Disparities in the availability of water between head-reach and tail-end farms and between large and
International Journal of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 6 small farms should be obviated by adoption of a rotational water distribution system and supply of water on a volumetric basis subject to certain ceilings and rational pricing; Concerted efforts should be made to ensure that the irrigation potential created is fully utilized. For this purpose, the command area development approach should be adopted in all irrigation projects.
Approach to Drought Management: The behaviour of the monsoon is usually erratic and uncertain in India. Kharif (summer crop) production depends on the quantum and distribution of rainfall. The behaviour of monsoon is broadly classified as: Normal season with normal onset, cessation and distribution of the monsoon; Delayed onset of the monsoon; Normal onset but early withdrawal of the monsoon; Normal onset and cessation but prolonged drought period in between (inter-spell dry period; Flood/excess rains; Uneven distribution of rain. The preparations for dealing with such situation, which is necessary to maintain from year to year, are: Early warning; Early response; An efficient intelligence system; Timely maintenance of the irrigation system and adoption of a crop stabilization strategy; An effective programme of relief works by advance shelf of projects of the works by different departments Pre-positioning of adequate foodstuff and their delivery; Alternate arrangements for drinking-water supply; Construction of deep wells and bore wells and repair of those which are defunct and continuous repair of hand pumps.
Initiative Taken for Drought Management: From 1900 to 2002, droughts in India resulted in 2 750 430 deaths and affected some 900 million people, apart from huge financial losses. It is the creeping effect of drought over long periods and its severity that sensitized the Government of India to treat the problem from several angles-scientific, technological, economic, social and environmental. Some of the initiatives taken for drought management by the Government are: Enhancement of the capabilities of long-range forecasts to climate modelling and weather forecasting; In 1989, the National Centre for Medium Range Weather Forecasting started to forecast weather on a medium-term basis (3-10 days in advance); Monitoring of storage position of reservoirs: 76 important reservoirs of the country having a total live storage capacity of 131.22 billion m3 are being monitored. A further 49 have also been identified for inclusion in the monitoring system, which will increase storage capacity of the monitored reservoirs to 156.69 billion m3, i.e. about 74 per cent of the total capacity of 213 billion m3 created so far; Efforts are under way to improve the efficiency of the irrigation system; The National Agricultural Drought Assessment and Monitoring System became operational in 1989; The National Centre for Disaster Management was set up in 1995 to undertake human-resource development, research, building a database and providing information services and documentation on disaster management; Many programmes to prevent/ mitigate drought in the long term; Supporting research to provide solutions to drought-related problems; Setting-up of a National Data Bank under the All India Co-ordinated Project on Agrometeorology at the Crop Research Institute for Dry Land Agriculture, Hyderabad; Setting-up of a National Disaster Management Authority. The new Drought Risk Management Programme under formulation aims to build on the previous Programme's experience to reduce the vulnerabilities of communities to drought through community-based approaches and appropriate risk management and better decision-support systems at state and district levels.
Approach to Flood Management: Approaches to dealing with floods may be any one or a combination of the following available options: Attempts to modify the flood Attempts to modify the susceptibility to flood damage Attempts to modify the loss burden Bearing the loss. The main thrust of the flood protection programme undertaken in India so far has been an attempt to modify the flood in the form of physical (structural) measures to prevent the floodwaters from reaching potential damage centres and modify susceptibility to flood damage through early warning systems.
International Journal of Engineering Research and Modern Education (IJERME) Impact The following structural measures are generally adopted for flood protection: Embankments, flood walls, sea walls Dams and reservoirs Natural detention basins Channel improvement Drainage improvement Diversion of flood waters. Of these measures, embankments are the most commonly undertaken in order to provide quick protection with locally available material and labour. The major embankment projects taken up after independence are on the rivers Kosi and Gandak (Bihar), Brahmaputra (Assam), Godavari and Krishna (Andhra Pradesh), Mahanadi, Brahmani, Baitarni and Subarnarekha (Orissa) and Tapi (Gujarat). These embankments play an important role in providing reasonable protection to vulnerable areas. Realizing the great potential of the reservoirs in impounding floods and regulating the flows downstream for flood moderation, flood control has been sought to be achieved as one of the objectives in multipurpose dams. Reservoirs with a specifically allocated flood cushion have been constructed on the Damodar system in Jharkhand and the Hirakud and Rengali dam in Orissa. However, many other large storage dams, e.g. Bhakra dam, without any earmarked flood storage, have also helped in flood moderation. During the post-independence period, multi-purpose projects such as the Damodar Valley Corporation (DVC) reservoirs, the Bhakra-Nangal project, Hirakud dam, Nagarjuna Sagar project etc., have been constructed to increase food production, energy generation, drinking-water supply, fisheries development, employment generation, flood moderation, etc. These large dams have played a significant role in reducing damage by way of flood moderation. One of the important flood moderation examples achieved by dams is that of Damodar Valley, where four reservoirs were constructed with flood management as one of the objectives. During the 2000 monsoon, DVC reservoirs saved the life and property of people from a possible disaster through flood moderation. Up to 2005, 34 398 km of new embankments and 51 318 km of drainage channels were constructed. In addition, 2400 town protection works were completed and 4 721 villages were raised above flood levels. Barring occasional breaches in embankments, these works gave reasonable protection to an area of some 16.5 million ha.
Non-Structural Measures: Non-structural measures include: Flood forecasting and warning Floodplain zoning Flood fighting Flood proofing Flood insurance. A brief description of the most important measure, i.e. flood forecasting, and the progress made so far is given below.
Flood Forecasting and Warning Network in India: Of all the non-structural measures for flood management which rely on the modification of susceptibility to flood damage, the one which is gaining increased/ sustained attention of planners and acceptance by the public is flood forecasting and warning, which enable forewarning as to when the river is going to use its floodplain, to what extent and for how long. As for the strategy of laying more emphasis on nonstructural measures, a nationwide flood forecasting and warning system has been established by the Central Water Commission. Flood forecasting and flood warning in India commenced in a small way in the year 1958 with the establishment of a unit in the Central Water Commission, New Delhi, for flood forecasting for the river Yamuna at Delhi. This has now grown to cover most of the flood-prone interstate river basins. The Central Water Commission is currently responsible for issuing flood forecasts at 173 stations, of which 145 are for river stage forecast and 28 for inflow forecast. On average, about 6 000 flood forecasts are issued every year with a maximum of 7 943 forecasts in 1998. The forecasts issued by the Central Water Commission have been consistent with about 96 per cent accuracy as per the present norms of the Central Water Commission. A forecast is considered to be reasonably accurate if the difference between forecast and corresponding observed level of the river lies within ±15 cm. In the case of inflow forecasts, variations within ±20 per cent are considered acceptable, as a result of which the flood-forecasting and warning services have rendered immense benefit to those in flood-prone areas.
Modernization of Flood Forecasting Services: The Central Water Commission is making a constant endeavour to update and modernize forecasting services on a continuous basis to make flood forecasts more accurate, effective and timely. Initiatives being taken for modernizing flood forecasting services are: International
Conclusion: As the world is speeding ahead in the field of artificial rains we INDIA an agricultural country which is mainly dependent on rains for water need to implement these modern techniques so that drought and floods could be avoided. As these methods are economical and non-hazardous they can be easily implemented and would help in serving the purpose of rains all over country and also drought affected areas. The potential technology of precipitation enhancement is very closely linked to water resources management. It is important that the users of this potential technology are integrated into programs at a very early stage in order to establish the requirements and economic viability of any program Future investigations thus should concentrate on establishing a physical hypothesis that incorporates all the major components of the precipitation formation processes in order to provide as sound a scientific basis as possible for estimating the magnitude of the expected effect. To attain this objective we will have to increase our understanding of the individual components of the precipitation formation process. Progress in understanding a single component of this process is intrinsically limited if an understanding of all the major components is not developed to comparable levels. It is very important not to make the same mistake with the new hygroscopic seeding method. Although very exciting and promising results have been obtained to date, some fundamental questions remain that need to be answered in order to provide a sound scientific basis for this technique. As mentioned in the introduction, water is becoming an ever more scarce and precious commodity around the world. The potential societal benefits of precipitation enhancement are therefore too important for us to ignore, and a coordinated strategy should be developed to provide a sound scientific basis for precipitation enhancement programs. With the new tools and techniques, the of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 3 Research Methodology: Changes on Various Aspects of Environments How Irrigation Has Effected Rainfall Patterns How Rainfall Can Be Made Possible By Various Artificial And Natural Methods Impact on Effectiveness Environment and Health International Journal
Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 7 Structural Measures:
Journal of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019 8 The establishment and modernization of the flood forecasting network, including inflow forecast through automated data collection and transmission; use of satellite-based communication systems through very small aperture terminals; and improvement of forecast formulation techniques using computer-based catchment models; Development of a decision-support system for flood forecasting and inundation forecast model for the Mahanadi basin and flash flood forecasting for Sutlej basin; Development of a real-time flood-forecasting system for the Brahmaputra and Barak basin, envisaging data collection through automatic sensors and transmission through satellite and forecast formulation using a computer-based mathematical model.Suggestions: The largest cloud seeding system is in the People's Republic of China. They believe that it increases the amount of rain over several increasingly arid regions, including its capital city, Beijing, by firing silver iodide rockets into the sky where rain is desired. There is even political strife caused by neighbouring regions that accuse each other of "stealing rain" using cloud seeding. About 24 countries currently practice weather modification operationally. China used cloud seeding in Beijing just before the 2008 Olympic Games in order to clear the air of pollution. In February 2009, China also blasted iodide sticks over Beijing to artificially induce snowfall after four months of drought, and blasted iodide sticks over other areas of northern China to increase snowfall. The snowfall in Beijing lasted for approximately three days and led to the closure of 12 main roads around Beijing. At the end of October 2009 Beijing claimed it had its earliest snowfall since 1987 due to cloud seeding. In India, cloud seeding operations were conducted during the years 1983, 1984-87, 1993-94 by Tamil Nadu Govt. due to severe drought in the years 2003 and 2004 Karnataka government initiated cloud seeding. Cloud seeding operations were also conducted in the same year through US-based Weather Modification Inc. in the state of Maharashtra. In 2008, there were plans for 12 districts of state of Andhra Pradesh. In Jakarta, cloud seeding was used to minimize flood risk in anticipation of heavy floods in 2013, according to the Agency for the Assessment and Application of Technology. In Southeast Asia, open burning haze that pollutes the regional environment. Cloud-seeding has been used to improve the air quality by encouraging rainfall. On 20 June 2013, Indonesia said it will begin cloud-seeding operations following reports from Singapore and Malaysia that smog caused by forest and bush fires in Sumatra have disrupted daily activities in the neighbouring countries. On 25 June 2013, hailstones were reported to have fallen over some parts of Singapore. Despite denials, some believe that the hailstones are the result of cloud seeding in Indonesia. In 2015 cloud seeding was done daily in Malaysia since the haze began in early-August. Thailand started a rain-making project in the late-1950s. Its first efforts scattered sea salt in the air to catch the humidity and dry ice to condense the humidity to form clouds. The project took about ten years of experiments and refinement. The first field operations began in 1969 above Khao Yai National Park. Since then rainmaking has been successfully applied throughout Thailand and neighbouring countries. On 12 October 2005 the European Patent Office granted to King Bhumibol Adulyadej the patent Weather modification by royal rainmaking technology.
International Journal of Engineering Research and Modern Education (IJERME) Impact Factor: 7.018, ISSN (Online): 2455 -4200 (www.rdmodernresearch.com) Volume 4, Issue 2, 2019
scientific community has an excellent opportunity to provide new insights, and to contribute substantially, to the benefit of water re-sources management around the globe
|
10.7554/elife.39876
|
cc-by|cc-by-nc-nd
| null | null |
openalex
|
Can a single regulatory sequence be shared by two genes undergoing functional divergence? Here we describe a single promiscuous enhancer within the Drosophila Antennapedia Complex, EO053, that directs aspects of the expression of two adjacent genes, pb (a Hox2 ortholog) and zen2 (a divergent Hox3 paralog), with disparate spatial and temporal expression patterns. We were unable to separate the pb-like and zen2-like specificities within EO053, and we identify sequences affecting both expression patterns. Importantly, deletion of EO053 affects expression of both genes as well, despite each gene having additional enhancers. We examine sequence conservation of EO053 within the Schizophora, and show that patterns of synteny between the Hox2 and Hox3 orthologs in Arthropods are consistent with a shared regulatory relationship extending prior to the Hox3/zen divergence. Thus, EO053 represents an example of two genes having evolved disparate outputs while utilizing this shared regulatory region.
2 .Introduction Changes in the expression specificity of genes involved in the development of multicellular organisms are implicated in modifications of form and function over evolution (Stern & Orgogozo, 2008; Wray, 2007; Rebeiz & Tsiantis, 2017; Rubinstein & de Souza, 2013) . To produce these distinct expression patterns, the promoters of many developmental genes are activated in specific spatiotemporal domains by one or more distal cis-regulatory sequences (Long, Prescott, & Wysocka, 2016; Levine, 2010) . Over the last three decades, two contrasting modes of promoter regulation by such sequences have emerged. Commonly, a gene specifically expressed in multiple diverse developmental contexts has distinct cis-regulatory sequences known as enhancers, each of which directs expression in a specific, limited subset of the overall context (Kuzin et al., 2012; Frost et al., 2017; Simonet, Bucay, Pitas, Lauer, & Taylor, 1991; MacNeill, French, Evans, Wessels, & Burch, 2000; Harding, Hoey, Warrior, & Levine, 1989) . In a second mode, multiple neighboring genes with overlapping expression domains can be controlled by a shared distal cis-regulatory region, referred to as a locus control region (LCR), that directs expression of the target genes in a common spatial and temporal pattern during development (Ahn, Mullan, & Krumlauf, 2014; Choi & Engel, 1988; Deschamps, 2007; Foley et al., 1994; Lehoczky, Williams, & Innis, 2004; Sharpe, Nonchev, Gould, Whiting, & Krumlauf, 1998; Spitz, Gonzalez, & Duboule, 2003; Tsai, Cooke, & Liebhaber, 2016; Jones, Monks, Liebhaber, & Cooke, 1995; Tsujimura, Chinen, & Kawamura, 2007; Mohrs et al., 2001) . These two modes of activation are not mutually exclusive, and genes 3 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint regulated by LCRs can also have their own independent enhancers (Deschamps, 2007; Jones et al., 1995) . Most experimental models for changes in patterns of gene expression have come from studies of specific enhancers. The developmental context of an enhancer's action is typically determined by the sequence-directed recruitment of specific DNA-binding transcription factors (Levine, 2010) . The specificity of an enhancer can be modified in evolution by DNA mutations affecting the complement of transcription factors recruited to the module (Glassford & Rebeiz, 2013; Rebeiz & Tsiantis, 2017; Stern & Frankel, 2013) . Thus, enhancers can acquire additional specificities that change the expression pattern of their target genes as long as the change is either not detrimental or accompanied by additional stabilizing mutations. Such models for enhancer evolution are often proposed in the context of "shadow enhancers", in which two enhancers regulating the same gene have overlapping and/or synergistic activity (Barolo, 2012; Perry, Boettiger, & Levine, 2011; Miller, Rebeiz, Atanasov, & Posakony, 2014; Cannavò et al., 2016; Stern & Frankel, 2013) . In this case, the partial redundancy between the two enhancers could buffer the effects of mutation and divergence of regulatory sequence (Payne & Wagner, 2015) . When an enhancer acquires multiple specificities, evolution can potentially lead to 1) loss of the newly acquired specificity (Jeong et al., 2008; Jeong, Rokas, & Carroll, 2006) , 2) loss of the original specificity, 3) complete loss of enhancer function, or 4) maintenance of the complex pattern. The latter two outcomes may be dependent upon the degree of use of 4 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint the same transcription factors for both specificities, as loss of binding sites for shared factors would affect both expression patterns (Rebeiz, Jikomes, Kassner, & Carroll, 2011) . In this work, we investigate an unusual case of complex expression through analysis of a 1.4 kb enhancer, referred to as EO053. We identified this region through the modENCODE effort, based upon detection of CBP binding only during embryonic stages ("Embryo Only 053") by chromatin immunoprecipitation (Negre et al., 2011) . We show that EO053 encodes complex spatiotemporal activity correlating with the evolutionary divergence in the expression and function of the two neighboring developmental genes under its regulatory influence. We present a distinctive mode of activation by EO053, in which each target gene utilizes EO053 for distinct spatiotemporal outputs. EO053 is located within an intron of the proboscipedia (pb) gene in Drosophila melanogaster, which encodes a homeodomain-containing transcription factor involved in patterning along the anteroposterior axis. pb is found in a complex of related homeobox (Hox) genes, a pattern common in metazoans (Lemons & McGinnis, 2006) . This collection of genes in D. melanogaster, referred to as the Antennapedia Complex (Antp-C), represents half of an ancestral Arthropod Hox gene complex that bifurcated within the Schizophora clade of flies into the Antp-C and Bithorax Complex (Bx-C), located 10,000 kb away (Negre & Ruiz, 2007) . Adjacent to pb, which is the Hox2 ortholog, are three genes derived from the ancestral Arthropod Hox3 gene: zerknüllt (zen), its duplicate zen2, and bicoid (bcd). At an early stage of insect evolution, the Hox3 ortholog (zen) diverged in both
5 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint expression and function away from anteroposterior patterning to specifying extraembryonic tissue at earlier stages in embryonic development (Hughes, Liu, & Kaufman, 2004) . More recently within Schizophoran flies, tandem duplications of zen produced zen2 and bcd (Stauber, Jäckle, & Schmidt-Ott, 1999; Stauber, Prell, & Schmidt-Ott, 2002; Negre et al., 2005) , the latter of which diverged further into a role as a morphogen specifying the anterior pole of the embryo (Driever & Nüsslein-Volhard, 1988; Struhl, Struhl, & Macdonald, 1989) . The insect radiation that followed the Hox3/zen divergence dates to the Devonian period (Misof et al., 2014) , implying that the regulatory changes in zen are roughly 400 million years old. Intriguingly, EO053 encodes expression patterns resembling both pb and its immediate upstream neighbor, zen2, suggesting this enhancer may regulate the expression of both genes even though they are activated in unrelated, non-overlapping tissues and developmental stages. Here we show that deletion of EO053 via CRISPR/Cas9 affects mRNA accumulation from both pb and zen2, indicating that indeed this enhancer is shared between these two genes. We also find that the sequences responsible for the pb-like and zen2-like expression patterns within EO053 are highly overlapping, and we identify nucleotide segments that alter both specificities. One such nucleotide block contains a conserved sequence existing prior to the Schizophoran zen-zen2 duplication, and we find that variants of this motif exhibit patterns of conservation within many of the major insect clades following the Hox3/zen divergence. Finally, we show that the pattern of synteny between zen and pb within the insects, and the lack of separation of these genes by translocation, is consistent with an ancient regulatory relationship between them, even in the face of disparately evolving specificities.
6 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint
Results
The EO053 enhancer specificities overlap the expression patterns of both pb and zen2 The location of EO053 within the large intron of pb suggested that pb itself may be the target of this enhancer (Fig. 1A ). Indeed, at embryonic stages when Pb protein is detected in maxillary and labial segments (Pultz, Diederich, Cribbs, & Kaufman, 1988) we find that EO053 drives GAL4 expression in these territories as well (Fig1E -G), although not in a pattern as expansive as that driven by the previously-studied pb regulatory region, 2.1 (Fig. 1A , (Kapoun & Kaufman, 1995a) ). Interestingly, in blastoderm stages EO053 also drives GAL4 expression dorsally along most of the length of the embryo (Fig. 1B, C ). Following gastrulation, GAL4 is detected in the amnioserosa (Fig. 1D ), a specificity derived from the earlier dorsal cell population (Hartenstein, 1995) . This pattern is not representative of pb, but rather mimics the expression of zen2 (Pultz et al., 1988) , the gene immediately upstream of pb (Fig. 1A ). This additional expression could simply represent a coincidental ectopic artifact of the precise genomic segment chosen for cloning the enhancer. However, the orthologous region from Drosophila virilis also encodes both expression specificities, reducing the likelihood of this being a chance occurrence (SFig 1A, C -C'').
7 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint
Figure 1. EO053 exhibits both zen2-like and pb-like expression patterns. A. Diagram of the pb (blue) and zen2 (yellow) genes and the locations of EO053 (black bar) and the 2.1 pb regulatory region (grey bar) (Kapoun & Kaufman, 1995a) . B-G. Expression of GAL4 mRNA expression by in situ hybridization in EO053>GAL4 embryos exhibits a pattern reminiscent of zen2 (Rushlow, Doyle, Hoey, & Levine, 1987) in early embryonic stages (B-D) and overlaps expression of pb (Pultz et al., 1988) in later stages (E-G). AS: amnioserosa. Md: mandibular segment. Mx: maxillary segment. Lb: labial segment. While numerous regulatory regions have been shown to serve more than one promoter (Choi & Engel, 1988; Deschamps, 2007; Foley et al., 1994; Lehoczky et al., 2004; Sharpe et al., 1998; Spitz et al., 2003; Tsai et al., 2016; Jones et al., 1995; Tsujimura et al., 2007; Mohrs et al., 2001) , these genes typically have expression specificities in common. EO053, then, may serve as an example of a regulatory region that serves more than one promoter but with each gene utilizing the region to generate a different specificity. We thus sought to determine how linked are these specificities and whether each gene indeed requires the EO053 region for expression.
The pb-like expression specificity derives from the central region of EO053 We first began analyzing EO053 under a simple model for encoding multiple specificities: Each expression pattern is dependent upon a separate subregion of the 1.4-kb EO053 sequence. We created a set of reporter constructs containing overlapping truncated 9 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint portions of EO053 (trunc1, trunc2, trunc3, trunc1-2, trunc2-3; Fig. 2 ). The central region, trunc2, drives both pb-like and zen2-like expression but not as robustly as the full EO053 construct (Fig. 2 E, F ). This region also drives ectopic expression in the ventral embryo at stage 10 (Fig. 2F ) and ectopic dorsal expression (amnioserosa or dorsal vessel) in late-stage embryos (Fig. 2G ). The right-most region, trunc3, also drives pb-like expression, though very weakly (Fig. 2I, J ). A construct that encompasses both the trunc2 and trunc3 regions drives reporter expression in a robust pb-like pattern that also lacks the ectopic activities seen with trunc2 alone (Fig. 2O, P ), suggesting that trunc3 contains elements that repress the late dorsal expression. This model is further supported by the trunc1-2 construct (removing the right-most portion of EO053) that also drives ectopic late dorsal expression (Fig. 2M ).
10 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The zen2-like and pb-like expression specificities are not easily separable While trunc2 retains some capacity to drive zen2-like expression (Fig. 2E ), it was only weakly detectable in a few early-gastrulation embryos (embryo in Fig. 2E is rare; most stage 5 embryos lack GAL4 expression). A construct including trunc2 and the left-most portion of EO053, trunc1-2, restores zen2-like expression (Fig. 2K ), yet the left-most portion alone, trunc1, fails to drive reporter expression at any stage (Fig. 2B-D ). Since these initial truncation constructs failed to reveal a region in EO053 responsible for the zen2-like expression, we designed a set of 10 smaller overlapping reporter constructs to locate the zen2-like activity (Fig. 3A ). None of these smaller fragments drive reporter expression in a zen2-like pattern (SFig. 2), while two fragments, truncF and truncG, drive reporter expression in a pb-like pattern (Fig. 3C, D, F, G ), consistent with their overlap with trunc2 (Fig. 3A ). Furthermore, we again observed late expression in the dorsal embryo driven by truncG (Fig. 3G ), refining the location of this ectopic activity seen neither with full-length EO053>GAL4 or endogenous pb or zen2 mRNA. These fragments allowed us to further define the domain sufficient to produce the pb-like pattern, and suggested that if the pb-like and zen2-like specificities were separable, the latter pattern would be localized to the left-most region of EO053 outside of truncF and truncG. Importantly, truncF-J, which lacks this left-most region, fails to drive zen2-like GAL4 expression (Fig. 3K ). A construct containing only this region, however, fails to drive strong zen2-like reporter expression (Fig. 3H ). This suggests that while the A-D region is necessary (but not sufficient) for the zen2-like pattern, the FG region likely also contains elements required for this specificity, in addition to being sufficient to drive pb-like expression. Consistent with this interpretation, a construct that deletes this region, truncDFG, lost both pb-like and zen2-like expression patterns (Fig. 3N-P ). Because the FG region is necessary for both the pb-like and zen2-like patterns, we sought an alternative approach to determine if the two patterns are indeed separable. In a series of eight constructs, we created successive 47-nt non-complementary transversion mutations along the length of the FG region to identify sequences required for either the pb-like or zen2-like patterns (Fig. 4 ). Interestingly, none of the 47-nt mutations could recapitulate the strong reduction of zen2-like activity seen in EO053DFG>GAL4. Two mutants, FG3 and FG7, either eliminate (FG3) or strongly reduce (FG7) the pb-like 15 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint expression pattern (Fig. 4F '-F'', J'-J''). Each of these mutants also affect the zen2-like pattern, though in opposite directions: FG3 causes an ectopic anterior expansion of the zen2-like pattern (Fig. 4F ), while FG7 reduces zen2-like expression (Fig. 4J ). FG1, FG2, FG4, FG6, and FG8 also reduce expression in the zen2-like pattern (Fig. 4D-K ). The reduced expression seen with the FG4 mutation results in dorsal stripes (Fig. 4G ), which were also observed with the insufficient truncA-D construct (Fig. 3H ). Together, these data suggest that while the pb-like pattern can be effectively localized to the FG region in EO053, the elements required for zen2-like expression are spread much more broadly throughout EO053 and are even linked to regions necessary for the pb-like pattern. A. Diagram of a series of 47-nt non-complementary transversion mutants generated within the FG region (FG1 -FG8, blue, with mutated segments shown in pink), the same region deleted in the truncDFG construct (green). B-K. GAL4 mRNA expression in blastodermstage embryos. Segment labels as in Figure 1 . zen2-like expression is absent in truncDFG (C); reduced in FG1 (D), FG2 (E), FG4 (G), FG6 (I), FG7 (J), and FG8 (K); and expanded anteriorly in FG3 (F: bracket). Inset in G is a dorsal view of an embryo exemplifying the pseudo-stripe pattern of GAL4 expression along the anteroposterior axis driven by the FG4 mutant reporter. B'-K''. GAL4 mRNA expression in stage 10-12 embryos (B'-K') and stage 13-16 embryos (B''-K''). pb-like expression is absent in truncDFG (C', C'') and FG3 (F', F''); and strongly reduced in FG7 (J', J'').
16
EO053 is required for the full expression of both pb and zen2 While the pb-and zen2-like specificities appear to be linked within the EO053 sequence, we sought to determine whether EO053 is functionally linked to either pb or zen2, or both. We thus generated via CRISPR/Cas9 a deletion of the EO053 region at the endogenous pb locus, pb M2:20 , and examined the effects upon both pb and zen2 mRNA accumulation (Fig. 5 ). Kapoun and Kaufman have shown pb mini-genes lacking large sections of the intron overlapping EO053 are capable of rescuing adult mouthparts-to-leg transformations in pb null flies and the 2.1 enhancer is required for rescue in the context of these small pb mini-genes (Kapoun & Kaufman, 1995a) . Thus, because the 2.1
18 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint enhancer is unaffected in pb M2:20 homozygous embryos, we were not surprised to observe detectable pb mRNA in these embryos. Kapoun and Kaufman also showed a 10.6 kb fragment-likely overlapping EO053 sequence-was able to drive LacZ expression in maxillary and, to a lesser extent, labial segments in embryos (Kapoun & Kaufman, 1995a) . While this is strikingly similar to the EO053 expression pattern we observe, it is possible additional pb enhancers outside of EO053 reside on this 10.6 kb fragment. Despite 2.1 and other potential pb enhancers remaining intact in pb M2:20 flies, in double-blind scoring of pb expression in parallel in situ hybridization experiments, we were able to observe a statistically significant reduction in pb mRNA accumulation in pb M2:20 embryos compared to w 1118 controls (Fig. 5C ). These data suggest that indeed EO053 serves a role as a dual enhancer of pb, likely operating in addition to the 2.1 and potentially other enhancers (Kapoun & Kaufman, 1995a) . We similarly anticipated that if EO053 regulates zen2 it may not act alone on this target either. Endogenous zen2 mRNA accumulation expands to the anterior and posterior poles of blastoderm-stage embryos (Fig. 5J ), a pattern that differs from expression driven by
21 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint EO053, which is absent from the poles (Fig. 1B ). While the regulation of zen2 expression has not yet been pursued, an investigation of the duplicated zen gene found that a reporter driven by the promoter-proximal region recapitulates endogenous zen gene expression (Doyle, Kraut, & Levine, 1989) . Guided by the zen2 inversion between D. melanogaster and D. virilis (SFig. 1D), we cloned the zen2 promoter-proximal region (zen2US) and found that it is indeed capable of driving reporter expression in a pattern that recapitulates the polar expansion of endogenous zen2 mRNA (Fig. 5H ). Intriguingly, the EO053 and zen2US patterns differ not only spatially but temporally, with zen2US driving reporter expression only until the completion of cellularization at stage 5 (Fig. 5H, I ), while EO053 is strongly active at this stage and continues to gastrulation (Fig. 1B-D ). Such a transition is also observed with endogenous zen2 mRNA: Early expression includes expression in polar regions as well as dorsally at stage 4 (Fig. 5J ), but following cellularization the mRNA is largely detectable only in dorsal-most cells and is absent from the anterior and posterior poles (Fig. 5K ). We find that this later accumulation of endogenous zen2 mRNA requires EO053, as pb M2:20 embryos largely fail to express zen2 beyond cellularization (Fig. 5M, M' ). Thus, EO053 appears to have dual roles in Drosophila embryogenesis, assisting other enhancers in blastoderm stages with zen2 expression and then with pb expression during later morphogenetic events (Fig. 7 ).
pb and zen genes remain syntenic despite the change in zen expression The zen, zen2, and bicoid (bcd) genes in Drosophila are derivatives of the ancestral Hox3 ortholog in basal arthropods, and have diverged in expression and function from the
22 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint ancient homeotic role. Why, then, do they remain at their ancestral genomic location within the Hox complex? Splits and inversions within the Hox complex are common in Schizophoran flies, suggesting loosened constraints on collinearity (Negre & Ruiz, 2007; Von Allmen et al., 1996) . The sharing of regulatory elements among members of the Hox complex has been a model to explain the persistent linkage of Hox genes in metazoan genomes (Spitz et al., 2003; Sharpe et al., 1998) , and the function of EO053 provides direct support for maintenance of an ancestral regulatory linkage as a contributing factor to persistence of a pb-zen linkage. While it is challenging to trace EO053 itself across evolution, patterns of synteny between pb and zen following the functional transition offer an opportunity to test such a model. Specifically, any translocation of a zen ortholog away from the pb ortholog would presumably not be favored if one or more regulatory elements are shared between the two genes. Indeed, examining available genomic scaffolds across 80 different Arthropods, we were unable to detect any translocation event that breaks the synteny between pb/Hox2 and zen/Hox3 (SFig. 3, SFig. 4). Furthermore, among the 66 species examined that evolved following the Hox3/zen divergence, only 3 species-all members of the Formicoidea-exhibit a change in synteny but only via the loss of the zen coding sequence (SFig. 3, SFig4D). In contrast, 3/14 species examined that predate the Hox3/zen divergence exhibit loss of Hox3 or pb-representing Crustacea, Myriapoda, and Chelicerata (SFig. 3, SFig. 4G, H) (Chipman et al., 2014; Grbić et al., 2011; Pace, Grbić, & Nagy, 2016; Kim et al., 2016; Kenny et al., 2014) .
23 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint
An EO053 motif important for both pband zen-like expression exhibits patterns of conservation within various clades Examining patterns of regulatory sequence conservation is a complementary approach to exploring the model of ancient, shared regulation as an explanation of the persistent pb/zen linkage. Sequence conservation makes possible the identification of EO053 throughout the Schizophora (Fig. 6A, B ). In particular, 33/36 nt are identical between D. melanogaster and Ceratitis capitata over the region including FG3 (Fig. 6C ), which we have shown to be required for the proper expression of both pb-and zen2-like specificities (Fig. 4F-F'' ). Outside of the Brachycera it is challenging to identify orthologous regulatory regions. Comparing D. melanogaster EO053 and pb intronic sequence from the mosquito Anopheles gambiae identified a 12-nt sequence from within the Schizophora 36-nt span (ATCATTAATCAT, henceforth referred to as "the EO053 motif", in green in Fig. 6B, C ) that is also found in the Anopheles intron (SFig. 4A). This sequence is similar to others that have been shown to be bound by Exd/Hox dimers (Bergson & McGinnis, 1990; Chan, Ryoo, Gould, Krumlauf, & Mann, 1997; Regulski, Dessain, McGinnis, & McGinnis, 1991; Rusch & Kaufman, 2000; Zeng, Pinsonneault, Gellon, McGinnis, & McGinnis, 1994) , and thus represents a plausible candidate for an ancient motif with regulatory function. The deepest we were able to identify a region orthologous to EO053 outside of Schizophora was in the assassin fly Proctacanthus coquilletti (Brachycera; Orthorrapha). This species contains a variant EO053 motif (ATCATAAATCAT) that could still mediate a Exd/Hox interaction (Slattery et al., 2011) . . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint We chose to examine the 80 Arthropod Hox2/3 regions for patterns consistent with an ancient regulatory function for this or similar sequences. from select Schizophoran flies, indicating additional sequences conserved in this region in these species (see also Supplemental Figure 4 ). 65/66 species that arose following the Hox3/zen divergence contain instances of the EO053 motif within the large introns between the YPWM-and homeodomain-encoding exons of pb (SFig. 4). The outlier Locusta migratoria scaffold containing the homeodomain lacks upstream motif instances but also lacks the upstream exon necessary to confirm motif absence (SFig. 4F). In species that arose prior to the Hox3/zen divergence, the Ixodes scapularis (Chelicerata) (SFig. 4H), Daphnia pulex (Crustacea) (SFig. 4G), and Orchesella cincta (Hexapoda) (SFig. 4F) pb orthologs lack intron motif instances. Despite a lack of direct evidence for which, if any, motifs in the Hox2/3 regions are functional outside of Schizophora, we nevertheless examined these genomic intervals for patterns of conservation. We identified in several major Arthropod clades conserved instances of the EO053 motif, based upon relative location and flanking sequences (SFig. 4, Supplemental File). Lepidoptera contain a conserved motif instance between pb and zen2 (Motif 2 in SFig. 4B, Supplemental File). Coleoptera contain a conserved mismatch upstream of the pb promoter (Motif 3 in SFig. 4C, Supplemental File), and several species contain conserved motifs within the pb intron (Motif 4) and upstream of the duplicated zen genes, including on an isolated scaffold harboring a fourth zen paralog in the Asian long-horned beetle A. glabripennis (Motif 5 in SFig. 4C, Supplemental File). Within Hymenoptera, the two basal Tenthredinoidea species have five conserved motif mismatches (Motifs 6 -10 in SFig. 4D, Supplemental File). Outside the Tenthredinoidea, all remaining species conserve a mismatch upstream of zen (Motif 11 in SFig. 4D, Supplemental File). Of particular note, this sequence is still present in the three Formicoidea that have lost the zen coding sequence. The Formicoidea also contain an intron motif specific to this clade (Motif 13 in SFig. 4D, Supplemental File), as well as another intron motif also conserved throughout the Aculeata (Motif 12 in SFig. 4D, Supplemental File). Several of the Hemiptera examined (excluding the Sternorrhyncha species A. pisum, D. citri, and B. tabaci) contain a conserved motif mismatch downstream of zen (Motif 14 in SFig. 4E, Supplemental File), and the two Dictyoptera species contain a conserved motif mismatch upstream of zen (Motif 15 in SFig. 4F, Supplemental File). Within the Chelicerata, gene duplication and loss presents interesting opportunities to examine motif instances. The multiple rounds of whole-genome duplication in the basal Limulus polyphemus led to three copies each of pb and Hox3, two of which have confirmed synteny. All three Hox3 paralogs have a motif mismatch downstream (Motif 16 in SFig. 4H, Supplemental File). At a similar position downstream of one of the pb paralogs is the same 12-nt motif, with a variant site at the same position downstream of a second pb. We found another curious parallel between one adjacent Limulus pb and Hox3: the same 12-mer mismatch motif is found in the introns of each gene, though upstream of the putative Hox3 coding sequence, at a similar distance from a separate motif instance (Motif 17 in SFig. 4H, Supplemental File). This same motif was also found upstream of the coding sequence of the third Limulus Hox3, again at a similar distance from another motif instance. We also found instances of these motifs in Arachnida, with a Hox3 ortholog in Centruroides exilicauda, Parasteatoda tepidariorum, and Stegodyphus mimosarium as well as a pb ortholog in Centruroides and Parasteatoda having the downstream motif (Motif 16); we also found a 12-mer matching the intron motif (Motif 17) in one of the pb paralogs in Stegodyphus (SFig. 4H,
28 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint Supplemental File). By contrast, we observed no conservation of a randomly generated 12mer (TGCATAAGTCGA, also allowing for a 1 bp mismatch) even when examining the Schizophoran regions, for example, with a single motif instance occurring every 325124 bp on average. Together our analysis indicates that while functional assumptions are limited to the Schizophora, sequences resembling the EO053 motif exhibit patterns of conservation across Arthropod clades within the Hox2/Hox3 genomic region, particularly within clades emerging after the Hox3/zen divergence.
29 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint
Discussion We have shown that the EO053 enhancer exhibits inherent regulatory complexity in two critical ways. First, it encodes more than one specificity, manifested by its ability to drive reporter gene expression in two distinct temporal and spatial patterns: the zen2-like dorsal expression in stage 5-7 blastoderm embryos and the pb-like maxillary and labial segment expression in stage 10-12 embryos. Second, each of these specificities is functionally linked to regulation of a separate target promoter. As such, this region serves as a curious contrast to existing models of complex regulatory output derived from examples of multiple independent enhancers working additively on a single target gene (Simonet et al., 1991; MacNeill et al., 2000; Harding et al., 1989) or control regions conferring common expression patterns upon multiple local target genes (Choi & Engel, 1988; Deschamps, 2007; Foley et al., 1994; Lehoczky et al., 2004; Sharpe et al., 1998; Spitz et al., 2003; Tsai et al., 2016; Jones et al., 1995; Tsujimura et al., 2007; Mohrs et al., 2001) .
Serving separate promoters Perhaps the most curious feature of EO053 is its requirement by distinct genes for the reliability (pb) or temporal progression (zen2) of their expression. Given its intronic location, we suggest that this regulatory arrangement would be mediated by a looping interaction between EO053 and each target promoter (Levine, Cattoglio, & Tjian, 2014; Matharu & Ahituv, 2015) . Such interactions are likely permitted by the distinct temporal activation profiles of each target gene, allowing the enhancer to separately engage only a single active promoter at a time (Fig. 7 ). In addition, we have gained insight into the 30 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint temporal dynamics with which EO053 operates on the zen2 locus, whereby the initial activation of zen2 expression is mediated by the promoter-proximal zen2US segment and then switches to control by EO053. The regulatory interactions between EO053 and both pb and zen2 are likely influenced by the overall regulatory architecture of the Antp Complex. Recent high-resolution analyses of topologically associated domains (TADs) suggest that pb, zen2, zen, and bcd all reside in a single TAD (Eagen, Aiden, & Kornberg, 2017; Stadler, Haines, & Eisen, 2017) , potentially biasing regulatory activities between these loci and separate from Dfd, which is a regulatory island (Stadler et al., 2017) . The three-dimensional architecture facilitating interactions between EO053 and its target promoters is likely mediated by Polycomb Response Elements (PREs) that have been mapped within the pb locus (Kapoun & Kaufman, 1995b; Negre et al., 2011; Stadler et al., 2017) and exhibit chromosomal
33 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint pairing experimentally (Kapoun & Kaufman, 1995b) . The observed establishment of Polycomb bodies in the nucleus at stage 5 (Cheutin & Cavalli, 2012 ) also correlates well with the temporal shift in regulation of zen2 from promoter-proximal to distal regulatory regions.
Distinct temporal and spatial specificities We have shown that the pb-like and zen2-like specificities overlap within the FG region of EO053. While the pb-like expression is largely restricted to this region, the zen2-like expression appears to be much more broadly extended throughout EO053. Moreover, within the FG region, mutation of FG3 or FG7 affects both specificities, suggesting the specificities may have one or more motifs in common in their regulatory logic. Such a motif could be bound by the same transcription factor in both settings or related factors with different temporal and/or spatial profiles. We raise the possibility that the conserved EO053 motif may represent such a site. First, its pattern of conservation and location within the functionally-important FG3 region suggests that this sequence itself may be required. Second, similar sequences have been identified as functionally relevant to the expression of Dfd (Chan et al., 1997; Zeng et al., 1994) (Bergson & McGinnis, 1990 ) (Regulski et al., 1991; Lou, Bergson, & McGinnis, 1995) and pb itself (Rusch & Kaufman, 2000) . Both of these examples involve regulation by Dfd, and indeed we also notice instances of this motif upstream of the Dfd orthologs in many of the species we have analyzed (SFig. 4), suggesting that Dfd auto-regulation within the arthropods may be ancient. Given the similar binding specificities of Hox proteins (Noyes et al., 2008; Berger 34 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint et al., 2008) , this site could be utilized by one or more of these proteins, even operating as a promiscuous auto-regulatory enhancer. This would be consistent with the demonstrated role of EO053 in the temporal dynamics of zen2 expression where its activity is preceded by zen2US activity, but would warrant future investigation in the dynamic use of 2.1 and EO053 by the pb promoter. We expect activation and specificity to also involve key regulators binding non-Hox motifs, which again could participate in one or both specificities. Why does neither pb nor zen2 mRNA expression reflect the complete regulatory capacity of EO053? A common feature of many enhancers is their ability to interact reliably with a heterologous promoter to drive reporter gene expression in a manner that not only largely recapitulates specificity encoded by the enhancer, but also represents a subset of the expression pattern of the endogenous gene. EO053, however, produces a pattern that would be considered an ectopic specificity relative to the pattern of either target gene (i.e. if EO053 were only a pb enhancer the blastoderm spaciotemporal activity is ectopic and the mouthpart activity does not recapitulate zen2 expression). Promoter selectivity/interpretation is a likely model to explain the different transcriptional outputs of the separate promoters that utilize EO053. The collection of core promoter elements at an individual promoter can bias promoter-enhancer compatibility (Butler & Kadonaga, 2001) , and the pb promoter itself is required for expression driven by certain enhancers (Kapoun & Kaufman, 1995a) . Replacing the promiscuous synthetic core promoter (Pfeiffer et al., 2008) in the pBPGUw vector used here with the minimal core promoter from either pb 35 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint (TATA box absent) or zen2 (TATA box present) had no effect on reporter gene expression (data not shown), which may suggest that promoter interpretation may require additional promoter-proximal elements to mediate the appropriate spatial/temporal output. It is additionally possible that an EO053-proximal element may facilitate appropriate promoter targeting and output (Chen, Lin, Smith, Lin, & Zhou, 2005; Zhou & Levine, 1999) .
Evolution and maintenance The stable collinearity of vertebrate Hox genes has been attributed to sharing of regulatory elements (Gould, Morrison, Sproat, White, & Krumlauf, 1997; Gérard et al., 1996; Sharpe et al., 1998) . Arthropods may not share this paradigm across the complete complex, as suggested by the occurrence of rearrangements (Pace et al., 2016; Faddeeva-Vakhrusheva et al., 2017; Wu et al., 2017) , inversions (Negre & Ruiz, 2007) , gene loss (Chipman et al., 2014; Grbić et al., 2011) , regulatory independence (Gellon & McGinnis, 1998; Shippy et al., 2008) , and local chromatin organization (Eagen et al., 2017; Stadler et al., 2017) . We provide evidence here for the possibility of limited shared regulation within an Arthropod Hox complex, between a true Hox (pb) and a neighboring Hox-derived gene (zen2) both sharing EO053. While we do not know how deeply this regulatory relationship extends, the organization of Hox complexes across the phylum exhibits features consistent with shared regulation. The strong synteny of pb and zen and reduced gene loss (a subset of ant genomes being the exception to date) suggests shared regulation may have existed from the point of Hox3/zen divergence, if not earlier. The most likely scenario based upon the available data would be a pb-Hox3 shared duplicate enhancer that drives a pattern of 36 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint expression common to both genes. Acquisition of a novel expression specificity by the shared enhancer would then be buffered by the additional independent regulatory sequences of each gene. Alternatively, the change in spatial/temporal expression of the shared enhancer might also impart selective pressure alleviated by differential interpretation of the enhancer by each gene, and/or functional divergence. Regardless of the mechanism, EO053 serves as an unusual example of an enhancer maintaining a promiscuous relationship with two distinct promoters even in the context of disparately evolving expression specificities.
37 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint
Materials and Methods
Reporter constructs The EO053 region was identified by enriched CBP binding in embryonic rather than later stages [i.e., "Embryo Only"(EO) (Negre et al., 2011) ]. It was amplified from genomic DNA using the primers EO053-f (CCCGGAGCGGCACAATTAGTCTTG) and EO053-r (CGGTAATGCTGAATGAACCTTTCAA). DFG and FG mutations were generated using overlap extension PCR (Ho, Hunt, Horton, Pullen, & Pease, 1989) . The DvEO053 primers were DvEO053-f (TGCCCTGGTTCTTTGGCTAACACG) and DvEO053-r (TTTCTTGTACATAATCGTTCTTGG). PCR products amplified from genomic DNA were cloned into pBPGUw (Pfeiffer et al., 2008) through an LR Clonase II (ThermoFisher-Waltham, MA) Gateway reaction from a pCR8/TOPO/GW intermediate. All variants were inserted upstream of the DSCP promoter in the same orientation with respect to EO053. zen2US was amplified using zen2US-f (TTATATACCCCAGAAGCCCTTCGTGACG) and zen2US-r (TGATGTGATGACACCAATTTATCTGAGC) and cloned as above.
Generation of pb M2:20 by CRISPR/Cas9 mutagenesis Preparation of guide RNA and Cas9 mRNA was done as described previously (Hwang et al., 2013) . The sequence for a guide RNA (GGAGTCGGTCGGACACAGAG) was cloned into DR274 (Addgene-Cambridge, MA; deposited by J Keith Joung) cut with Bsa I. Sequence-verified clones were linearized with Dra I and 1 μg transcribed using the MAXIscript T7 kit (ThermoFisher). Cas9 mRNA was transcribed using the mMESSAGE mMachine T7 kit, using 1 μg MLM3613 (Addgene; deposited by J Keith Joung) linearized
38 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint with Pme I. RNAs were precipitated according to the manufacturer's instructions and resuspended in injection buffer. The final injection cocktail for injecting w 1118 embryos contained 900 ng/μL Cas9 mRNA and 100 ng/μL EO053 guide RNA. Injected flies were crossed to w;;TM2/TM6C individually, and then 20 F1 males from each injected fly were crossed individually to w;;TM2/TM6C. After viable larvae were detected, the F1 males were removed from the vials and pooled into groups of 4 for gDNA extraction and PCR screening for deletion. F2 vials from positive pools were screened and sequenced to uncover the pb M2:20 1255-bp deletion.
In situ hybridization The GAL4 digoxygenin probe was prepared as previously described (Negre et al., 2011; Pfeiffer et al., 2008) . The large exons from pb and zen2 were amplified from genomic DNA and cloned into pGEM-T (Promega-Madison, WI). Linearized plasmids served as template for in vitro transcription of digoxygenin-labeled RNA probes as described (Kosman et al., 2004) using T7 RNA polymerase (Promega) and DIG-UTP RNA labeling mix (Roche). Embryo in situ hybridizations using GAL4, pb, or zen2 digoxygenin probes were performed as previously described (Negre et al., 2011) (Reeves & Posakony, 2005) . GAL4 in situ hybridizations with related mutant constructs were performed in parallel batches and representative images presented. pb in situ hybridization image scoring 39 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint Following in situ hybrization and mounting of w 1118 and pb M2:20 embryos in parallel, images were collected of lateral views of stage 10 embryos. Filenames of experimental and control in situ hybridization images were gathered, randomly shuffled, and renamed for double-blind scoring on a 0 -3 qualitative scale. After scoring, images were reassigned based upon genotype and statistically analyzed.
zen2 in situ hybridization image scoring Following in situ hybridization and mounting of w 1118 and pb M2:20 embryos in parallel, slides were manually screened for dorsal visibility at stage 4 or stage 5 (staging based upon cellularization under DIC optics). The in situ hybridization signal at these stages was scored as "dorsal-weak," "dorsal-strong," "poles+dorsal," "poles-strong," "poles-weak," or "no expression." In Figure 5 , "dorsal-weak" and "dorsal-strong" represent the "dorsal" category, and "poles+dorsal," "poles-strong," and "poles-weak" represent the "polar" category, to distinguish between EO053-like and zen2US-like expression.
Scaffold analysis We queried genomic scaffolds of sequenced arthropods by BLAST using Drosophila melanogaster amino acid sequence for Pb, Zen2, Zen, or Dfd, as well as orthologous sequences from other annotated species. Scaffolds or accession numbers were obtained from http://metazoa.ensembl.org/, http://hymenopteragenome.org/, http://www.vectorbase.org/, http://i5k.nal.usda.gov/, http://www.ncbi.nlm.nih.gov/genbank/, http://genome.wustl.edu/, 40 . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 doi: bioRxiv preprint http://www.collembolomics.nl/collembolomics/. Gene structures were inferred from either annotations or database gene predictions. For some species, only the homeodomain sequence for each gene was determined and located. In some cases (e.g., Dendroctonus ponderosae), the pb and zen homeodomains are encoded on separate scaffolds but the zen scaffold includes near one end a YPWM-encoding ORF that by BLAST is most similar to pb, suggesting that the scaffolds are likely adjacent.
Gene diagrams and motif analysis Each sequence was opened in GenePalette (Rebeiz & Posakony, 2004; Smith, Posakony, & Rebeiz, 2017) , and the location of each gene identified by either GenBank or manual annotation. Sequences were searched for instances of ATCATTAATCAT (allowing for 1-bp mismatch), "ATTAAT" (ATCATTAAT or ATTAATCAT), and "YPWM" (TAYCCNTGGATG). Instances found at similar relative locations in related species were analyzed for similarity in both core and flanking sequence to suggest orthology within clades. All figure gene diagrams were generated by creating a postscript export from GenePalette and compiling/editing in Adobe Illustrator. Figure 2 . 2 Figure 2. pb-like expression driven by EO053 can be localized to a central region of the
Figure 3 . 3 Figure 3. zen2-like expression driven by EO053 requires the central region of the
Figure 4 . 4 Figure 4. Mutation of specific nucleotide stretches in the FG region of EO053 can affect
Figure 5 . 5 Figure 5. Deletion of EO053 affects mRNA accumulation from both pb and zen2.
Figure 6 . 6 Figure 6. Conservation of EO053 sequences within the Schizophora.
Figure 7 . 7 Figure 7. Possible model for EO053 regulation of both zen2 and pb.
International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under bioRxiv preprint bioRxiv preprint doi: doi: It is made available under The copyright holder for this preprint (which was not . CC-BY-NC-ND 4.0 The copyright holder for this preprint (which was not this version posted July 7, 2018. ; https://doi.org/10.1101/364216 this version posted July 7, 2018. ; https://doi.org/10.1101/364216
|
10.3390/s17020292
|
cc-by
| null | null |
openalex
|
Calorimetric biochemical measurements offer various advantages such as low waste, low cost, low sample consumption, short operating time, and labor-savings. Multichannel calorimeters can enhance the possibility of performing higher-throughput biochemical measurements. An enthalpy sensor (ES) array is a key device in multichannel calorimeters. Most ES arrays use Wheatstone bridge amplifiers to condition the sensor signals, but such an approach is only suitable for null detection and low resistance sensors. To overcome these limitations, we have developed a multichannel calorimetric simultaneous assay (MCSA) platform. An adjustable microampere constant-current (AMCC) source was designed for exciting the ES array using a microampere current loop measurement circuit topology. The MCSA platform comprises a measurement unit, which contains a multichannel calorimeter and an automatic simultaneous injector, and a signal processing unit, which contains multiple ES signal conditioners and a data processor. This study focused on the construction of the MCSA platform; in particular, construction of the measurement circuit and calorimeter array in a single block. The performance of the platform, including current stability, temperature sensitivity and heat sensitivity, was evaluated. The sensor response time and calorimeter constants were given. The capability of the platform to detect relative enzyme activity was also demonstrated. The experimental results show that the proposed MCSA is a flexible and powerful biochemical measurement device with higher throughput than existing alternatives.Introduction Catalase is a common enzyme present in nearly all living organisms exposed to oxygen, and it is a crucial enzyme in protecting cells from oxidative damage engendered by reactive oxygen species. Catalase activity has been demonstrated to be involved in the development of human type 2 diabetes [1, 2] . Erythrocyte catalase is the principal regulator of hydrogen peroxide metabolism; any inherited or acquired deficiencies in erythrocyte catalase may thus cause the hydrogen peroxide concentration in the body to increase, which can have both toxic and physiological effects. Blood catalase activity is generally measured using a clinical spectrophotometric assay. The blood catalase activity of a reference range of 1753 patients was discovered to be 80.3-146.3 units/μL . The activity assay of this enzyme and other enzymes is usually challenging and time-consuming to quantify using chemical analysis [3, 4] . Catalase catalyzes the decomposition of hydrogen peroxide into oxygen and water and generates a molar enthalpy of -98.2 kJ/mol , given by the expression: H 2 O 2 Catalase ----→ 1 2 O 2 + H 2 O + -98.2 kJ mol (1) Calorimetric measurements are principally enthalpy measurements. The heat change caused by a biochemical or physical reaction is measured using the temperature change of a solution in a calorimeter. Microcalorimeters are currently used to detect and characterize substrate concentrations, enzyme activity, and protein-ligand structures . The enthalpy sensor (ES) array is a crucial device in microcalorimeters used in the physical, chemical, and biochemical fields, especially for the high-throughput assays required in modern science. Weber and Salemme reported that high-throughput calorimeters can be incorporated into the drug discovery process . Enthalpy can be measured in calorimeters using three quantities: temperature change, power compensation, and heat conduction. The enthalpy of protein-small molecular and protein-protein interactions has been detected to be between -90.8 and 7.95 kJ/mol in some studies [6, 7] . An enthalpy sensor with microjoule sensitivity can be used to calorimetrically measure micromoles of protein. In recent years, microfabricated enthalpy arrays have been developed using n + amorphous silicon and vanadium oxide [9, 10] thermistors for detecting the heat involved in biochemical reactions. The major merits of these enthalpy arrays are that they are very small and have a high heat sensitivity level for calorimetric assays. Each detector of these enthalpy arrays applies a Wheatstone bridge, and a reaction volume of only 500 nL (two 250 nL drops) is required. The enthalpy arrays employ an on-chip electrostatically merging and mixing mechanism using voltages of 100 and 180 V to commence the calorimetric reaction. The temperature coefficients of resistance (TCR) and noise equivalent temperature differences of n + amorphous silicon and vanadium oxide thermistor were determined to be 2.8%/ C and 2.7%/ C and 50-100 and 30 μK root mean square (rms), respectively. The detectable temperature change, detectable heat, and thermal dissipation times were reported to be 500 and 30 μK, approximately 1 and 46 μJ, and approximately 1.3 and 2.0 s, respectively. A Deerac spot-on liquid handling system was used to deposit drops on these enthalpy arrays. Another chip calorimeter using a multichannel three-dimensional thermopile for basic applications was reported . This chip calorimeter was evaluated using Joule heating and TRIS/HCl reactions, and its temperature sensitivity and heat power sensitivity were determined to be 78 μV/K and 63.2 mV/W, respectively. Microcalorimeters used for biochemical reactions need to be able to measure temperature changes ranging from tens of microKelvins and hundreds of milliKelvins. A thermopile-based chip calorimeter cannot measure real temperatures but only the change in voltage that occurs with a change in heat. Such enthalpy arrays and chip calorimeters involve a chip with a thermal sensor array, but are not true multichannel calorimeters. These devices use a commercial nanovoltmeter or Wheatstone bridge amplifier to measure the output voltage of a thermopile or thermistor array. The Wheatstone bridge circuit's ability to measure small changes in electrical resistance has been long proven, especially when a strain gauge or a Pt-100 probe is employed . Therefore, the Wheatstone bridge circuit has been used in enthalpy arrays. However, this is only suitable for null detection and low-resistance sensors, as reported by Anderson . The principle of thermodynamics states that the heat evolved during a biochemical reaction at constant pressure is equal to the change in enthalpy. Heat is a form of energy that causes temperature changes and can be detected by various thermal sensors such as thermocouples, negative temperature coefficient (NTC) thermistors, platinum resistance temperature detectors, and semiconductor sensors . For the detection of microJoule-level heat, an NTC thermistor appears to be a suitable choice because of its high sensitivity, short response time, small size, and low manufacturing costs. However, the relationship between the temperature and resistance of an NTC thermistor is nonlinear. To achieve high sensing performance, it is necessary to consider array-excited methods, nonlinear calibration equations, and signal-amplifying methods when using the signal conditioner of an NTC thermistor-based ES array. Previously, our group developed a prototype single-channel calorimetric assay device using a single NTC-thermistor-based ES, 200-μL acrylic reaction cell and a homemade half-bridge Wheatstone amplifier . This simple calorimeter had a calibrated temperature accuracy of ± 0.001 C and a residual H 2 O 2 detection limit of 53 ppm. The relationship between temperature change and H 2 O 2 concentration was obtained as 0.0079 C/mM when using catalase as catalyst. To yield higher accuracy with a smaller amount excitation compared to using the Wheatstone bridge while accomplishing additional measurement functions, we have designed an adjustable microampere constant current (MACC) looped-type source such that multichannel ES arrays can be implemented in MCSA platform. Such structures have not been reported by others. The contributions and aims of this work are: (1) design a novel seven channel microcalorimeter array and a new detachable reaction vessel to perform multichannel simultaneous calorimetric assays; (2) design a novel adjustable microampere constant current source to excite a seven NTC-thermistor-based ES array in a single current loop to overcome the limitations of Wheatstone bridges; (3) design a novel dual-work mode multichannel signal processing circuit for ES arrays to support high precision temperature measurements and a voltage differential measurement mode; (4) preliminarily evaluate relative enzyme activity using multichannel simultaneous calorimetric measurements.
System Design In this study, we designed a novel MCSA platform for simultaneously detecting multiple enzyme activities in a biochemical assay. To the best of our knowledge, no similar design has been previously reported. The MCSA platform comprises a measurement unit and a signal processing unit.
Measurement Unit Figure 1a presents a complete three-dimensional exploded view of the interior and exterior of the MCSA platform measurement unit. The components of the platform are outlined as : (1) bottom of the unit, composed of a highly thermally conductive metal (Al); (2) Al wall, (3) Al calorimeter holder, (4) sensor plug (4.9 mm diameter, 23 mm high), composed of polytetra-fluoroethene (PTFE), a poorly thermally conductive polymer; (5) PTFE down cell of the calorimetric vessel (4.9 mm inner diameter, 12 mm outer diameter, 12 mm high); ( 6 ) polyethylene (PE) isolating film (10 μm thick), (7) PTFE up cell of the calorimetric vessel (4.9 mm inner diameter, 12 mm outer diameter, 22 mm high); ( 8 ) polypropylene (PP) pipe knife with 45 blade; (9) sensors' wire connectors; (10) top cover made of another poorly conductive polymer, polymethylmethacrylate (PMMA); (11) stepping motor (ZKM2283-02TE2, Zi Sheng Motion Technologies Inc., Taichung, Taiwan) for injection; (12) injecting actuator of the stepping motor screw shaft; (13) PMMA simultaneously injecting plate; (14) up-position limit switch of the injecting plate; (15) down-position limit switch of the injecting plate; and (16) slip shaft (PSFGN-L100-F20-P4, Misumi Taiwan Cor., Taipei, Taiwan) for the injecting plate, linear bearing, and limit switches. The measurement unit consists of a multichannel calorimeter (I) and an automatic multichannel simultaneous injector (II). Figure 1b displays the detailed arrangement of the sensor plug and chip heater in a detachable reaction vessel when electrical calibration. The calorimeter includes seven channels, where six channels are used for biochemical reactions and one channel serves as a thermal reference. All ES arrays are connected through a wire connector and wired by seven pairs of 90-cm-length insulated Cu cable (core resistance: 0.169 Ω ± 0.001 Ω per 90 cm) to the AMCC looped-type source in the signal processing unit. A plate-type automatic injector was designed to execute simultaneous multichannel injection in the measurement unit of the MCSA platform. The operation of the injector is trigged by a manual/automatic injection controller (not shown), consisting of a motor driver, a microcontroller, and a power supplier. A thermal sink was required to stabilize the temperature of the microcalorimeter array block. Accordingly, our design involves a circulation water bath that comprises an immersed water pump, a thermoelectric cooling (TEC) device, a controllable pulse width modulation (PWM) power regulator, a 12 VDC power supplier, a thermostatic water tank with cover, and a proportional-integral-derivative (PID) temperature controller. The water bath maintains the temperature of the microcalorimeter array block at 298.13 ± 0.1 K. The baseline temperature fluctuation of the detachable thermal reference reaction vessels was determined to be approximately ±0.002 K in within an hour.
Signal Processing Unit The signal processing unit is mainly consists of a sensor signal conditioner (I) and a data processor (II) as illustrated in Figure 2 . The sensor signal conditioner comprises a sensor array, AMCC source, radio frequency (RF) interferometer, and 10-fold-gain instrument amplifier (IA) with passive low-pass-filter in the firstand second-stage circuits. A pair switch enables switching between "mK" and "microJ" work mode. The "mK" work mode measures of milliKelvin temperature changes, whereas the "microJ" work mode measures microJoule voltage changes. The sensor array consists of seven NTC-thermistor-based ESs and one current sensor. The sensor array is connected in series with the AMCC source in a loop circuit. Seven commercial available interchangeable ultraprecision NTC-thermistor-based ESs, (PR103J2, accuracy ±0.05 K, R298.13K = 10 kΩ, β = 3892 K, Dissipation Constant [DC] = 1 mW/K; US Sensor Corp., Orange, CA, USA), are installed in seven sensor plug (Figure 1 ) of the multichannel microcalorimeter array and connected separately by seven 90-cm-length dual-core-insulated Cu cables to the terminal of handmade circuit board. The current sensor is a highly accurate low-temperature coefficient resistor (10 kΩ, 0.01%, TC5; Caddock Electronics Inc., Roseburg, OR, USA). Installed directly on terminal of hand-welded
Signal Processing Unit The signal processing unit is mainly consists of a sensor signal conditioner (I) and a data processor (II) as illustrated in Figure 2 . The sensor signal conditioner comprises a sensor array, AMCC source, radio frequency (RF) interferometer, and 10-fold-gain instrument amplifier (IA) with passive low-pass-filter in the first-and second-stage circuits. A pair switch enables switching between "mK" and "microJ" work mode. The "mK" work mode measures of milliKelvin temperature changes, whereas the "microJ" work mode measures microJoule voltage changes. The sensor array consists of seven NTC-thermistor-based ESs and one current sensor. The sensor array is connected in series with the AMCC source in a loop circuit. Seven commercial available interchangeable ultraprecision NTC-thermistor-based ESs, (PR103J2, accuracy ±0.05 K, R 298.13K = 10 kΩ, β = 3892 K, Dissipation Constant [DC] = 1 mW/K; US Sensor Corp., Orange, CA, USA), are installed in seven sensor plug (Figure 1 ) of the multichannel microcalorimeter array and connected separately by seven 90-cm-length dual-core-insulated Cu cables to the terminal of handmade circuit board. The current sensor is a highly accurate low-temperature coefficient resistor (10 kΩ, 0.01%, TC5; Caddock Electronics Inc., Roseburg, OR, USA). Installed directly on terminal of hand-welded circuit board, it serves as a standard resistor to measure the constant current flowing in the sensor loop in real time. circuit board, it serves as a standard resistor to measure the constant current flowing in the sensor loop in real time. In this study, we designed a novel AMCC source to excite the sensor array. A transistor NPN (Q2) works as an open-collector constant-current source that is compensated by a single transistor PNP (Q1). The circuit is presented in Figure 2 , and the collector current is given by Q2 Q2 Q2 C B I β I (2) The current ICQ2 is related to the base current of Q1 as 2 2 2 1 1 [ ( 1 ) ] CQ Q R Q BQ I β I β I , (3) where IBQ1 is determined by 1 1 1 1 EB BQ BQ BQ V V V I R (4) IR2 is the current of R2, V + is the power of +5 V dc, VEB1 is the potential between the emitter and the base of Q1, VBQ1 is the base bias voltage of Q1 and RBQ1 is the base resistance of Q1. According to Equations ( 2 ) and (3), ICQ2 is high when the base bias voltage of Q1 (VBQ1) is high, because of IQB1 is low. When VBQ1 is kept constant, ICQ2 is constant. In this study, we designed a novel AMCC source to excite the sensor array. A transistor NPN (Q2) works as an open-collector constant-current source that is compensated by a single transistor PNP (Q1). The circuit is presented in Figure 2 , and the collector current is given by I CQ2 = β Q2 × I BQ2 (2) The current I CQ2 is related to the base current of Q1 as I CQ2 = β Q2 × [I R2 -(β Q1 + 1)I BQ1 ], (3) where I BQ1 is determined by I BQ1 = V + -V EB1 -V BQ1 R BQ1 (4) I R2 is the current of R2, V + is the power of +5 V dc, V EB1 is the potential between the emitter and the base of Q1, V BQ1 is the base bias voltage of Q1 and R BQ1 is the base resistance of Q1. According to Equations ( 2 ) and (3), I CQ2 is high when the base bias voltage of Q1 (V BQ1 ) is high, because of I QB1 is low. When V BQ1 is kept constant, I CQ2 is constant. To reduce RF noise, an RF interferometer (RFI) was used in front of the inputs of differential IA by using a differential low-pass filter . In our design, the -3 dB differential bandwidth and common-mode bandwidth were determined to be 7.5 and 159.1 Hz, respectively. The data processor consists of an ADC module and a microprocessor unit (MPU). All conditioned sensor signals are sent to the ADC module (LTC2418CGN; Linear Technology Company, Milpitas, CA, USA) to digitize the eight amplified sensor signals. In our study, the reference voltage (V REF ) was supplied from VCC source (+5 V dc). The full-scale bipolar input range was from -0.5 to +0.5 V REF . A user-programmable single-chip MPU (Linduino One; Linear Technology Company, Milpitas, CA, USA) module was used to digitize the amplified multiple ES array analog signals, perform digital voltage-to-temperature conversion, and transmit data to a personal computer. Windows-based data-logging software (MakerPlot; Selmaware Inc., Rocklin, CA, USA) was used to monitor and record data from the data processing unit. Microsoft Excel was used to retrieve data and perform curve-fitting.
Experiments
Performance Evaluation of AMCC Source To minimize the self-heating effect of the NTC-thermistor-based ES array to a level of 1 μW, we self-designed an AMCC source. The 1-μW level guaranteed that the temperature change caused by the self-heating effect of the sensor would be less than 1 mK. The power of self-heating (P SH ) is defined as the product by the square of the excited current (I exc ) and the resistance of the sensor (R sensor ) at a working temperature 298.15 K: P sh = I exc 2 × R sensor . The self-heating-induced temperature change (T SH ) is defined as the division of the power of self-heating by the dissipation constant (DC): T SH = P SH /DC. When the AMCC source was adjusted by less than 8 μA, the T SH was controlled at less than 640 μK. The accuracy of the AMCC source was evaluated by the following tests. The correlation between the input voltage and output current of the AMCC source in the range 0-50 μA was determined through real measurements and software simulation (LTspice IV; Linear Technology Company, Milpitas, CA, USA). Tests of the static and dynamic stability level of the AMCC source were also performed. The static stability evaluation was conducted by inserting current probes between the current sensor (Rref) and NPN collector terminal (Q2). A 6.5-digit-precision multifunction meter (DMM-4040; Tektronix, Beaverton, OR, USA) in the range of 3-10 μA for every μA was employed in the static stability test. The dynamic stability evaluation was conducted by allowing the MCSA platform to measure real-time data in the range of 2-8 μA for every μA over a period of 1 h. Microsoft Excel was used to perform the statistical analysis of the dynamic stability test.
Electrical Calibration of the MCSA Platform Twelve miniature microwatt-level chip heaters were used to electrically calibrate the proposed MCSA platform. A 1.25 V alkaline battery (AA size; Panasonic, Osaka, Japan) was used as the power source of the heat pulse generator. To improve control precision during a heat pulse, a digital timer (H5CX-A-N, Omron, Osaka, Japan) was used and set at 0.01-s resolution. During each electrical calibration, six miniature chip heaters were individually immersed in the six calorimeter reaction vessels. The vessels were filled with 200 μL of pure water and ESs were installed at the bottom-center of each vessel (Figure 1 ). Each heater chip was soldered with a pair of AWG30 silver-coated copper wires (R30-1000; OK Industrial, Ishpeming, MI, USA) and connected to the timer. The Joule heating for the electrical calibration process was controlled using a manual trigger switch. When the timer relay was switched, the battery current flowed through all six miniature chip heaters and produced a heat pulse in each calorimeter reaction vessel. To test the sensor's application range, six miniature heaters with heating rates (resistance value) of 78 (20 kΩ), 156 (10 kΩ), 313 (4.99 kΩ), 781 (2.0 kΩ), 1562 (1.0 kΩ), and 3110 (499 Ω) μW (±1%) were used in the electrical calibration process. The sensor array was excited with a current of 8 μA. The potential difference between two terminals of each sensor was amplified with the first-stage 10-fold-gain IA (i.e., using the "mK" mode). The baseline noise levels were measured after initial thermal equilibrium was attained, and statistical analysis was performed, calculating the maximum, minimum, mean, and standard deviation in Kalvin. To test the sensors' precision of measurement, miniature heaters with heating rates (resistance value) of 78 (20 kΩ), 97 (16 kΩ), 104 (15 kΩ), 111 (14 kΩ), 120 (13 kΩ), and 130 (12 kΩ) μW (±1%) were used in the electrical calibration process. The sensor array was excited with currents of 3, 5, and 8 μA, and the potential difference between each sensor was amplified with two-stage 10-fold-gain IA (i.e., using the "microJ" mode). The baseline noise levels were measured after initial thermal equilibrium was attained, and statistical analysis was performed, calculating the maximum, minimum, mean, standard deviation, and peak-to-peak voltage in μV and equivalent Kelvin.
Calorimetric Enzyme Activity Assay The evolution of heat is one of the general features associated with exothermic bio-chemical reaction. The total heat evolution under adiabatic conditions is proportional to the molar enthalpy change (-∆H) and to the total number of product molecules (n p ) created in the reaction. The resulting temperature change (∆T) is inversely proportional to the total heat capacity of the system (C SYS ) including the heat capacity of the solvent. This relationship can be described by ∆T = (n p (-∆H)/C SYS (5) The change in temperature can then be measured using the ES in the calorimeter to determine system's heat capacity, substrate concentration, protein concentration, and relative enzyme activity during biochemical reactions. In this study, we demonstrated the performance of the proposed MCSA platform by observing one exothermic enzyme reaction and measuring its relative activity. Purified catalase (CAT), from bovine liver (C3155; Sigma-Aldrich Cor., St. Louis, MO, USA), was used instead human blood. Relative catalase activity was examined using a modified Nelson's method before the calorimetric assay. Briefly, phosphate buffer (0.05 mol/L at pH 7.0) was prepared and used for dilution and reactions. Hydrogen peroxide (0.02 mol/L) was prepared and used as substrate. A typical test used 120 μL of hydrogen peroxide in each calorimetric vessel down cell; catalase assay solution was diluted to 1/1, 1/2, 1/3, 1/4, 1/5 and 1/6 fold and filled into the up cell during an assay. The real concentration of hydrogen peroxide substrate solution was calibrated using a spectrophotometer (USB4000, Ocean Optics, Inc., Dunedin, FL, USA) operating at λ = 240 nm; the concentration was determined every 10 s for 5 min at 25 C. The actual H 2 O 2 concentration was calculated by using Beer's Law (ε mM = 0.0436) : [H 2 O 2 ](mM) = A 240 /0.0436, (6) where A 240 is the absorbance of substrate solution at 240 nm. The actual activity of the diluted catalase solution was calibrated according to the rate of decomposition of hydrogen peroxide, which is proportional to the reduction of the absorbance of hydrogen peroxide at 240 nm. Hydrogen peroxide concentrations were then calculated using the measured absorbance of the solution at a wavelength of 240 nm, with the optical path length being 1 cm and the molar extinction coefficient was 0.0436 mM/cm
Results and Discussion
Accuracy and Stability of AMCC Source Real data were measured by using a 6.5-digit-precision multifunction meter (DMM-4040, Tektronix), whereas simulated data were obtained using LTspice IV software (Linear Technology Corporation). As illustrated in Figure 3 , both the measured (black) and LTspice IV simulated (red) output currents of the proposed AMCC source were linear when the base bias voltage of Q1 (V BQ1 ) varied from 0.07 to 2.518 V. The difference in the coefficients of determination between the two regressions lines was relatively small. Such small differences are mainly caused by the insertion of current meter probes serially between the current sensor (Rref) and the NPN collector (Q2) when measuring a real current.
Results and Discussion
Accuracy and Stability of AMCC Source Real data were measured by using a 6.5-digit-precision multifunction meter (DMM-4040, Tektronix), whereas simulated data were obtained using LTspice IV software (Linear Technology Corporation). As illustrated in Figure 3 , both the measured (black) and LTspice IV simulated (red) output currents of the proposed AMCC source were linear when the base bias voltage of Q1 (VBQ1) varied from 0.07 to 2.518 V. The difference in the coefficients of determination between the two regressions lines was relatively small. Such small differences are mainly caused by the insertion of current meter probes serially between the current sensor (Rref) and the NPN collector (Q2) when measuring a real current. The accuracy and stability of the current directly affect the performance of the proposed ES array. Static stability analyses were conducted using the statistic function built into the 6.5-digit-precision multifunction meter (DMM-4040) with respect to 3000 measured data points. The results indicate that the when the output current (ISET) was adjusted from 2.998 to 10.00 μA, the overall standard deviation (ISD) of the output currents was within 1.027-1.304 nA, implying that the overall error percentage ranged from 0.03% to 0.01%. The dynamic stability of the current output was tested over a period of 1 h. Dynamic stability analyses were performed using online real-time measurement, wherein all ES arrays included current sensor are connected in current loop and the MCSA platform was operated. The measured current data were collected using the software MakerPlot. The statistical analyses were executed using Microsoft Excel. The overall standard deviation (ISD) of the output currents were within 0.271-1.652 nA, which indicates that the overall error percentage varies from 0.01% to 0.02%, when the output current (ISET) was adjusted from 2.00 to 8.00 μA. These results indicated that the lower loop current has high stability than did the higher loop current during real-time operation.
Performance Evaluation of the Proposed MCSA Platform
Electrical Calibration of Application Range Using "mK" Mode The sensitivity of the proposed MCSA platform was evaluated using different Joule heating strengths. The measurement conditions used in sensitivity evaluation are outlined as follows: loop current = 8 μA; heater power voltage = 1.2468 V; heat pulse time = 20 s; gain = 10-folds; amplifier = first-stage 10-fold differential IA; heater = six miniature chip heater; power range = 0.078-3.110 mW. The temperature response curves are presented in Figure 4 . The calorimeters have time constants of The accuracy and stability of the current directly affect the performance of the proposed ES array. Static stability analyses were conducted using the statistic function built into the 6.5-digit-precision multifunction meter (DMM-4040) with respect to 3000 measured data points. The results indicate that the when the output current (I SET ) was adjusted from 2.998 to 10.00 μA, the overall standard deviation (I SD ) of the output currents was within 1.027-1.304 nA, implying that the overall error percentage ranged from 0.03% to 0.01%. The dynamic stability of the current output was tested over a period of 1 h. Dynamic stability analyses were performed using online real-time measurement, wherein all ES arrays included current sensor are connected in current loop and the MCSA platform was operated. The measured current data were collected using the software MakerPlot. The statistical analyses were executed using Microsoft Excel. The overall standard deviation (I SD ) of the output currents were within 0.271-1.652 nA, which indicates that the overall error percentage varies from 0.01% to 0.02%, when the output current (I SET ) was adjusted from 2.00 to 8.00 μA. These results indicated that the lower loop current has high stability than did the higher loop current during real-time operation.
Performance Evaluation of the Proposed MCSA Platform
Electrical Calibration of Application Range Using "mK" Mode The sensitivity of the proposed MCSA platform was evaluated using different Joule heating strengths. The measurement conditions used in sensitivity evaluation are outlined as follows: loop current = 8 μA; heater power voltage = 1.2468 V; heat pulse time = 20 s; gain = 10-folds; amplifier = first-stage 10-fold differential IA; heater = six miniature chip heater; power range = 0.078-3.110 mW. The temperature response curves are presented in Figure 4 . The calorimeters have time constants of about 200 s. The ES array sensed heat pulses from miniature chip heaters in reaction vessels. A high peak indicates that the electrical heat pulse was terminated because the timer relay shut off the power from the battery; thus, no more heat could accumulate in the detachable reaction vessels. Since the reaction vessels were installed in aluminum holders and fixed on an aluminum bottom plate that was in thermal equilibrium with the thermal sink. Thus, the accumulated heat in the reaction vessels was quickly lost through the holder because of its high heat transfer coefficient. This caused the temperature to decrease in the reaction vessel. Finally, the total change in temperature returned to zero, indicating that the calorimeters were thermally balanced. Both the bottom plate and thermal sink were in thermal equilibrium. The initial thermal equilibrium baseline noise levels of six microcalorimeters were statistically analyzed. The average baseline temperature noise was between 76 and 204 μK (standard deviation = 121-172 μK), equivalent to average voltage noise between 0.16 and 8.93 μV (standard deviation = 4.03-6.63 μV), respectively. Sensors 2017, 17, 292 9 of 16 about 200 s. The ES array sensed heat pulses from miniature chip heaters in reaction vessels. A high peak indicates that the electrical heat pulse was terminated because the timer relay shut off the power from the battery; thus, no more heat could accumulate in the detachable reaction vessels. Since the reaction vessels were installed in aluminum holders and fixed on an aluminum bottom plate that was in thermal equilibrium with the thermal sink. Thus, the accumulated heat in the reaction vessels was quickly lost through the holder because of its high heat transfer coefficient. This caused the temperature to decrease in the reaction vessel. Finally, the total change in temperature returned to zero, indicating that the calorimeters were thermally balanced. Both the bottom plate and thermal sink were in thermal equilibrium. The initial thermal equilibrium baseline noise levels of six microcalorimeters were statistically analyzed. The average baseline temperature noise was between 76 and 204 μK (standard deviation = 121-172 μK), equivalent to average voltage noise between 0.16 and 8.93 μV (standard deviation = 4.03-6.63 μV), respectively. From the temperature-time curve (Figure 4 ) obtained from each individual heat pulse, a tangent was drawn for the initial part of the curve (peak high temperature change/the heat pulse time). The obtained temperature change rate (K/s) with six chip heater power settings were graphed in Figure 5a . The regression curve of the rate of temperature change versus the Joule heating power (0.000078-0.00311 W) is displayed in Figure 5b . The calibration constant was defined as the inverse of the slope of the regression, i.e., 0.76 J/K; which corresponded to temperature change sensitivity with respect to voltage, i.e., 25.84 K/V. The obtained data indicates that the detection limit of the proposed platform was 1.558 mJ in "mK" mode (loop current = 8 μA), equivalent to a temperature change rate of 0.1 mK/s. To evaluate the percentage of heat loss preliminarily, the relationship between the input-heat and measured-heat was calculated. The measured-heat was calculated by P Δ Δ H m C T , ( 8 ) when m = 0.18 g is the mass of pure water in the reaction vessel, Cp = 4.186 J/gK is the specific heat capacity of pure water, and ∆T is the measured temperature change (taken from Figure 4 ). The correlation between the calculated data and the input electrical heat was found to be linear, with a slope of 0.999. However, the calorimeter array did lose a non-negligible amount of heat. During the electrical calibration process, approximately 200 μL of pure water was added to each reaction vessel. Thus, the calorimeter array had a heat loss of at least approximately 10%. From the temperature-time curve (Figure 4 ) obtained from each individual heat pulse, a tangent was drawn for the initial part of the curve (peak high temperature change/the heat pulse time). The obtained temperature change rate (K/s) with six chip heater power settings were graphed in Figure 5a . The regression curve of the rate of temperature change versus the Joule heating power (0.000078-0.00311 W) is displayed in Figure 5b . The calibration constant was defined as the inverse of the slope of the regression, i.e., 0.76 J/K; which corresponded to temperature change sensitivity with respect to voltage, i.e., 25.84 K/V. The obtained data indicates that the detection limit of the proposed platform was 1.558 mJ in "mK" mode (loop current = 8 μA), equivalent to a temperature change rate of 0.1 mK/s. To evaluate the percentage of heat loss preliminarily, the relationship between the input-heat and measured-heat was calculated. The measured-heat was calculated by ∆H = m × C P × ∆T, (8) when m = 0.18 g is the mass of pure water in the reaction vessel, C p = 4.186 J/gK is the specific heat capacity of pure water, and ∆T is the measured temperature change (taken from Figure 4 ). The correlation between the calculated data and the input electrical heat was found to be linear, with a slope of 0.999. However, the calorimeter array did lose a non-negligible amount of heat. During the electrical calibration process, approximately 200 μL of pure water was added to each reaction vessel. Thus, the calorimeter array had a heat loss of at least approximately 10%.
Sensitivity Tests Under Various Loop Currents Using "microJ" Mode A higher exciting loop current produced higher potential in the thermistor-based sensor array. Moreover, higher amplification gain increased the sensitivity of the sensor array as well as the noise level. The system response between different loop currents were used was investigated using Joule heating in a narrow range. The measurement conditions used in sensitivity test are outlined as follows: loop current = 3, 5, and 8 μA; heater power voltage = 1.25 V; heat pulse time = 30 s; gain = 100-fold; amplifier = dual stage of 10-fold differential IA; heater = six miniature chip heater; power range = 0.000078-0.00013 W. Test results when loop currents of 3, 5, and 8 μA were used, with six runs performed for each current, and are presented in Figure 6 . The correlation between the responses of the proposed MCSA platform and the power was linear. The detectable heat pulse levels of the platform when worked in "microJ" mode and the loop current of 3, 5 and 8 μA were used were 2.5348, 4.5107 and 7.8651 V/J, respectively. These data indicated that the minimum detectable heat was 1.424 μJ under 8 μA exciting current in "microJ" mode, with the standard deviation of the ADC being 11.2 μV, which corresponds to 0.00002 C. In Figure 6 , various loop currents lead to different heat pulse sensitivity. In real applications, such an arrangement would not be affected, because the experiments were used to evaluate the performances of the MCSA platform at various loop currents. The results support to use a low loop current to lower self-heating effects in the future.
Sensitivity Tests Under Various Loop Currents Using "microJ" Mode A higher exciting loop current produced higher potential in the thermistor-based sensor array. Moreover, higher amplification gain increased the sensitivity of the sensor array as well as the noise level. The system response between different loop currents were used was investigated using Joule heating in a narrow range. The measurement conditions used in sensitivity test are outlined as follows: loop current = 3, 5, and 8 μA; heater power voltage = 1.25 V; heat pulse time = 30 s; gain = 100-fold; amplifier = dual stage of 10-fold differential IA; heater = six miniature chip heater; power range = 0.000078-0.00013 W. Test results when loop currents of 3, 5, and 8 μA were used, with six runs performed for each current, and are presented in Figure 6 . The correlation between the responses of the proposed MCSA platform and the power was linear. The detectable heat pulse levels of the platform when worked in "microJ" mode and the loop current of 3, 5 and 8 μA were used were 2.5348, 4.5107 and 7.8651 V/J, respectively. These data indicated that the minimum detectable heat was 1.424 μJ under 8 μA exciting current in "microJ" mode, with the standard deviation of the ADC being 11.2 μV, which corresponds to 0.00002 C. In Figure 6 , various loop currents lead to different heat pulse sensitivity. In real applications, such an arrangement would not be affected, because the experiments were used to evaluate the performances of the MCSA platform at various loop currents. The results support to use a low loop current to lower self-heating effects in the future. Figure 7 illustrates the highest baseline noise level in one of six microcalorimeter arrays. For each exciting current used, six repeat measurements with equal power resulted in similar responses. However, the noise level was substantial in the test cases of specific channel. Moreover, the baseline noise varied with the measuring time. These offsets were caused by the irregular geometric thermal Figure 7 illustrates the highest baseline noise level in one of six microcalorimeter arrays. For each exciting current used, six repeat measurements with equal power resulted in similar responses. However, the noise level was substantial in the test cases of specific channel. Moreover, the baseline noise varied with the measuring time. These offsets were caused by the irregular geometric thermal distribution of the microcalorimeter array and the temperature drift of the thermal sink. This effect was particularly marked for the data in Figure 7c . In a multichannel calorimeter array design, the geometric thermal distribution should be considered carefully to minimize the long-term baseline temperature offset and drift. The magnitude of the baseline noise was dependent on: (1) the design of the electrical circuit; (2) the layout quality of the printed circuit board (PCB); and (3) the gain of signal amplifier. The baseline noise basically was discovered to originate from the 60-Hz power line after an observation was made using a spectrum analyzer. Furthermore, the baseline noise did not decrease when the loop current was increased but the electrical noise continued in specific channels. Care must be taken when designing and laying out prototype PCBs so that baseline noise is minimized. distribution of the microcalorimeter array and the temperature drift of the thermal sink. This effect was particularly marked for the data in Figure 7c . In a multichannel calorimeter array design, the geometric thermal distribution should be considered carefully to minimize the long-term baseline temperature offset and drift. The magnitude of the baseline noise was dependent on: (1) the design of the electrical circuit; (2) the layout quality of the printed circuit board (PCB); and (3) the gain of signal amplifier. The baseline noise basically was discovered to originate from the 60-Hz power line after an observation was made using a spectrum analyzer. Furthermore, the baseline noise did not decrease when the loop current was increased but the electrical noise continued in specific channels. Care must be taken when designing and laying out prototype PCBs so that baseline noise is minimized. The details of the initial thermal-balance baseline noise between the six ESs, as illustrated in Figure 7 , were statistically analyzed. The maximum noise, minimum noise, average noise, standard deviation of the average noise, peak-to-peak noise, and conversion of the baseline peak-to-peak noise from voltage into temperature were calculated. The peak-to-peak baseline noise was converted from voltage to temperature by multiplying the (p-p) by 25.84 K/V, and this was the temperature change sensitivity in the "mK" mode. The highest peak-to-peak noise was in the channel of 97-μW. When the exciting currents of 3, 5 and 8 μA were applied, the peak-to-peak noise levels were 23.83, 27.67 and 20.58 μV, respectively, equivalent to 615.77, 714.99 and 531.79 μK temperature change, respectively.
Catalase Activity Detection To demonstrate the capability of the proposed MCSA platform in preliminary biochemical assay, we designed an assay experiment for detecting enzyme activities. In this assay, 2.4 μmol (20 mM × 120 μL) calibrated hydrogen peroxide solution was used as a substrate and calibrated 120-μL diluted catalase solution was used as enzyme. The A 240 of the original hydrogen peroxide substrate solution was measured as 0.428. This corresponded to a real concentration of hydrogen peroxide substrate for the calorimetric assay of 20 mM. The catalase reaction rate, as measured by the slope of the A 240 curve, was obtained as 0.1696 per min. The real initial catalase activity was calculated to be 592.66 units/mL. Six diluted catalase enzyme solutions with activities of 0.296, 0.247, 0.198, 0.148, 0.099, and 0.049 units/μL, which were calibrated by spectrophotometric method, were used for the calorimetric assay experiments. Figure 8 presents the preliminary results of the relative enzyme activity detection of calibrated catalase. In Figure 8a shows a thermogram of the experimental test when a solution with a relative enzyme activity of 0.198 units/μL was introduced into the lower substrate cell at 82 s. The thermal sink remained at 298.13 K as the datum line. Both the upper cell with the enzyme solution and the lower cell with the substrate one were thermally balanced with the surroundings. We did not monitor the temperature in the up cell. The thermally unbalanced upper cell solution might be a source of disturbance in the injection, and the effect might be negligible as shown in the figure. The temperature fluctuated by approximately ±0.001 K before the reaction due to the use of an accurate thermistor and a stable thermal sink, and the temperature fluctuation was minor as compared to the temperature elevation during the reaction. Enzyme activities could be quantified on the MCSA platform by measuring the heat rate of an exothermic reaction. Assuming that the heat capacity of the sample solutions is constant, and that the reaction system is adiabatic at the very short initial stage, the heat rate could be linearly expressed in a temperature change rate. Taking the enzyme activity of 0.198 units/μL as an example. Figure 8a clearly shows that the temperature of the biocatalytic process increased linearly after the onset of the reaction. The temperature change rate of the reaction for this sample (0.198 units/μL) was estimated to be around 0.013 K/s. A similar calculation was applied to five other samples. The temperature change rates against the six enzyme activities (units/μL) are plotted in Figure 8b . The results indicate that the relationship between temperature change rate (K/s) and enzyme activity (units/μL) is linear with a slope of 0.066 K/s/unit/μL, calculated by linear regression. The reciprocal of the slope would be the measurement constant for the catalase activity on the MCSA platform. In the report of Góth [1, 3] , the blood catalase activity of 1753 patients as determined by a spectrophotometric assay was 80.3-146.3 units/μL, where one unit of catalase decomposes 1 μmol of hydrogen peroxide in 1 min under these conditions and it is related to 1 mL of whole blood. When the assay is conducted with a spectrophotometric method, every test requires four preparations and four measurements, once for a sample and three times for a blank. When our proposed MCSA assay platform is used, every test requires only a 1-μL blood sample and 120 μL of buffer.
Discussion The temperature resolution of the presented MCSA platform was up to ±10 μK (predicted calibration error between 20 and 30 C = -20 μK ± 10 μK), which is improvement on the device reported in our previous work, which had a resolution of ±1000 μK (calibrated by the primary SPRT 25.5 Ω thermometer). The temperature change and detectable Joule heating of the MCSA platform were 38 mV/K and 7.865 V/J, respectively. From previous reports, the temperature and heat or power sensitivity of the Ni/Au thermocouple were 0.078 mV/K and 63.2 mV/W, respectively ; those of an n + amorphous silicon (n + ) thermistor were 500 μK and 0.5 K/J (TCR: 2.8%/C), respectively ; and that of a vanadium oxide (VOx) thermistor was 890 K/W (TCR: 2.7%/C) [9, 10] . In recent years, microfabricated enthalpy arrays have been developed using n + and VOx [9,10] thermistors for detecting the heat involved in biochemical reactions. These enthalpy arrays are extremely small and have high heat sensitivity for calorimetric assays. However, Wheatstone bridges are used in each detector of those enthalpy arrays. Therefore, till now microfabricated enthalpy arrays still cannot provide multichannel simultaneous assay measurements. In our study, a microampere constant-current loop was employed to excite seven NTC-thermistor-based sensor arrays in the MCSA platform. The reaction volume in the n + and VOx enthalpy array was only 500 nL (two 250 nL drops) and was dropped on the detector region, whereas two drops of 120 μL were injected separately in the up cell and down cell of the detachable reaction vessel in the MCSA platform.
Discussion The temperature resolution of the presented MCSA platform was up to ±10 μK (predicted calibration error between 20 and 30 C = -20 μK ± 10 μK), which is improvement on the device reported in our previous work, which had a resolution of ±1000 μK (calibrated by the primary SPRT 25.5 Ω thermometer). The temperature change and detectable Joule heating of the MCSA platform were 38 mV/K and 7.865 V/J, respectively. From previous reports, the temperature and heat or power sensitivity of the Ni/Au thermocouple were 0.078 mV/K and 63.2 mV/W, respectively ; those of an n + amorphous silicon (n + ) thermistor were 500 μK and 0.5 K/J (TCR: 2.8%/ C), respectively ; and that of a vanadium oxide (VOx) thermistor was 890 K/W (TCR: 2.7%/ C) [9, 10] . In recent years, microfabricated enthalpy arrays have been developed using n + and VOx [9,10] thermistors for detecting the heat involved in biochemical reactions. These enthalpy arrays are extremely small and have high heat sensitivity for calorimetric assays. However, Wheatstone bridges are used in each detector of those enthalpy arrays. Therefore, till now microfabricated enthalpy arrays still cannot provide multichannel simultaneous assay measurements. In our study, a microampere constant-current loop was employed to excite seven NTC-thermistor-based sensor arrays in the MCSA platform. The reaction volume in the n + and VOx enthalpy array was only 500 nL (two 250 nL drops) and was dropped on the detector region, whereas two drops of 120 μL were injected separately in the up cell and down cell of the detachable reaction vessel in the MCSA platform. An automated simultaneous plate-type injector was used in the proposed MCSA platform for mixing and initializing the calorimetric reaction, whereas an on-chip electrostatic merging and mixing mechanism using voltages of 100 and 180 V was employed in the n + and VOx enthalpy arrays to initiate the calorimetric reaction. Both could induce a drift of the thermal signal in real operation. The TCR of the MCSA thermistor was 4.4%/ C, whereas those of the n + and VOx thermistors were 2.8 and 2.7%/ C, respectively. In addition, the differential temperature noise levels in n + and VOx thermistors were 50-100 and 30 μK rms, respectively; moreover, that of the MCSA platform was less than 204 μK on average. The detectable temperature change of n + and VOx thermistors were 500 and 30 μK, respectively, whereas that of the MCSA platform was 100 μK/s. The detectable heat in the n + and VOx thermistors and the MCSA platform in microJ were 1, 46, and 1.424 μJ, respectively. Furthermore, the thermal dissipation time in n + and VOx thermistors were approximately 1.3 and 2.0 s, respectively, whereas that of the MCSA's thermistor was approximately 7.72 s (response time was 1.25 s in water). An expensive Deerac spot-on liquid handling system was used to deposit the drops on the n + and VOx enthalpy arrays, whereas a cheap manual electrical pipette was used in the MCSA platform.
Conclusions We have developed and demonstrated a microampere constant-current-loop-excited multiple NTC-thermistor-based ES array that incorporates a multichannel microcalorimeter array in a single block. The predicted calibration temperature measurement error achieved by the MCSA platform was -20 μK ± 10 μK (between 20 and 30 C). The newly PNP-regulated NPN-transistor-based AMCC source provided a stable current between 2 and 8 μA with an error of 0.02%. An 8-μA constant current supported the self-heating of the NTC-thermistor-based ES within a temperature level of 640 μK. When operating in "mK" mode, the proposed MCSA platform had a calibration constant of 0.76 J/K and the Joule heating heat pulse detection limit was discovered to be 1.56 mJ. When the proposed MCSA platform was operated in "microJ" mode, its detectable Joule heating heat pulse was increased to 1.424 μJ. The proposed MCSA platform was also successful in the detecting purified catalase enzyme activities. The relationship between the temperature change rate (K/s) and relative catalase activity (units/μL) was linear. Catalase as well as other enzyme activities could be estimated by measuring the temperature change rate on the MCSA platform. The experimental results show that the proposed MCSA is a flexible and powerful biochemical measurement device with higher throughput. Sensors 2017, 17 , 292 4 of 16 aFigure 1 . 17161 Figure 1. (a) The three-dimensional exploded view of the measurement unit of the proposed multichannel calorimetric simultaneous assay platform; only one of seven detachable reaction vessels is displayed; (b) Detailed arrangement of sensor plugs and chip heater in detachable reaction vessel during electrical calibration.
Figure 1 . 1 Figure 1. (a) The three-dimensional exploded view of the measurement unit of the proposed multichannel calorimetric simultaneous assay platform; only one of seven detachable reaction vessels is displayed; (b) Detailed arrangement of sensor plugs and chip heater in detachable reaction vessel during electrical calibration.
Figure 2 . 2 Figure 2. Schematic diagram of the signal processing unit including multiple ES signal conditioners (I) and a data processor (II).
Figure 2 . 2 Figure 2. Schematic diagram of the signal processing unit including multiple ES signal conditioners (I) and a data processor (II).
Figure 3 . 3 Figure 3. Relationship between input voltage and output current of the proposed transistors-based adjustable microampere constant-current source.
Figure 3 . 3 Figure 3. Relationship between input voltage and output current of the proposed transistors-based adjustable microampere constant-current source.
Figure 4 . 4 Figure 4. Joule heating pulses (20 s) response of the proposed multichannel calorimetric simultaneous assay platform in "mK" mode (10-fold gain IA amplifier). Baseline temperature drift rate was 4 μK/s between measurements.
Figure 4 . 4 Figure 4. Joule heating pulses (20 s) response of the proposed multichannel calorimetric simultaneous assay platform in "mK" mode (10-fold gain IA amplifier). Baseline temperature drift rate was 4 μK/s between measurements.
Figure 5 . 5 Figure 5. Electrical calibration of the proposed multichannel calorimetric simultaneous assay platform. (a) Temperature-time plot in the present of various Joule heating power of the proposed MCSA platform; (b) Regression curve between the temperature change rate and Joule heating power.
Figure 5 . 5 Figure 5. Electrical calibration of the proposed multichannel calorimetric simultaneous assay platform. (a) Temperature-time plot in the present of various Joule heating power of the proposed MCSA platform; (b) Regression curve between the temperature change rate and Joule heating power.
Figure 6 . 6 Figure 6. Heat pulse sensitivity for the proposed multichannel calorimetric simultaneous assay platform when various loop currents and six miniature chip heaters. Test results with respect to three loop currents: (a) 3 μA; (b) 5 μA; (c) 8 μA (Joule heating pulse time: 30 s).
Figure 6 . 6 Figure 6. Heat pulse sensitivity for the proposed multichannel calorimetric simultaneous assay platform when various loop currents and six miniature chip heaters. Test results with respect to three loop currents: (a) 3 μA; (b) 5 μA; (c) 8 μA (Joule heating pulse time: 30 s).
Figure 7 . 7 Figure 7. Three observed heat pulse response curves of the proposed multichannel calorimetric simultaneous assay platform under test loop currents of (a) 3; (b) 5; (c) 8 μA.
Figure 7 . 7 Figure 7. Three observed heat pulse response curves of the proposed multichannel calorimetric simultaneous assay platform under test loop currents of (a) 3; (b) 5; (c) 8 μA.
Figure 8 . 8 Figure 8. Detection of purified catalase, from bovine liver (C3155). (a) Thermogram example of catalase activity 0.198 units/μL; (b) Temperature change rates between different enzyme activities.
Figure 8 . 8 Figure 8. Detection of purified catalase, from bovine liver (C3155). (a) Thermogram example of catalase activity 0.198 units/μL; (b) Temperature change rates between different enzyme activities.
), and 0.0436 is the molar extinction coefficient of H 2 O 2 (mM/cm). The calibrated catalase activities for calorimetric test were 0.296, 0.247, 0.198, 0.148, 0.099, and 0.049 units/μL. . Catalase activity was calculated by units/mL = [∆A 240 /min(blank) -∆A 240 /min(cat)] × d × l 0.0436 × V (7) where ∆A 240 /min(blank) is the slope of reduction rate of the blank H 2 O 2 solution at 240 nm per min, ∆A 240 /min(cat) is the slope of reduction rate of H 2 O 2 and catalase mixing solution at 240 nm per min, d is dilution of the catalase solution, l is optical path length in cm, V is the reaction solution sample volume (mL
|
10.1051/matecconf/201925805030
|
cc-by
| null | null |
openalex
|
Barite is a non-metallic mineral, which composed of Barium Sulfate (BaSO4), has specific gravity about 4.5. It can be used for high density concrete as a shielding against gamma-ray. This paper presents the use of barite as concrete aggregates to block gamma-ray radiation. Two concrete grades fc'25 and fc'35 were prepared. The effect of barite to the attenuation coefficient of samples was studied by replacing coarse aggregate with barite and replacing both coarse and fine aggregates with barite. The results show that the protection ability of the concrete using barite aggregates subjected to gamma-ray is better than those of concrete using barite as coarse aggregate and the normal one. The attenuation coefficient of concrete fc'25 with barite as aggregates and concrete with barite as coarse aggregate is 0.294 cm -1 and 0.230 cm -1 , respectively; The attenuation coefficient of concrete fc'35 with barite as aggregates and concrete with barite as coarse aggregate is 0.304 cm -1 and 0.271 cm -1 , respectively; While the attenuation coefficient of normal concrete fc' 25 and fc'35 is 0.205 cm -1 and 0.225 cm -1 , respectively. The average density of normal concrete fc' 25 and fc'35 was 2252 kg/m 3 and 2323 kg/m 3 , 3004 kg/m 3 and 3064 kg/m 3 for concrete with barite as coarse aggregate, and 3461 kN/m 3 and 3464 kg/m 3 for concrete using barite for its aggregates.Introduction Lead is metallic material that commonly used as a shielding against gamma-ray radiation emission . However lead is very expensive so that it is not economical to be installed on large scale. According to The National Council on Radiation Protection and Measurements , only materials which have high Specific gravity and atomic number exhibit capability to absorb radiation emission. Several materials that have high specific gravity (or density) are barite, magnetite, hematite, and other type of iron ores. Therefore these high density materials can be used as aggregates for concrete that specifically produced for radiation protection . The minimum unit weight () of concrete that can be used to block gamma-rays and x-rays radiation emission is 3.2 ton/m 3 . With specific gravity of 4.5, barite is very common and effectively to be used as high density concrete to absorb radiation emission [5, 6] . Esen and Yimazer stated that there was a significant relationship among the increase of barite content, density of the concrete, and radiation permeability. As the specimen thickness and barite ratio increase, radiation permeability decreases and becomes zero after a certain thickness. The capability of materials to absorb radiation emission commonly presented as linear attenuation coefficient (). Barite is a non-metallic mineral that composed of barium sulfate (BaSO4) exhibits specific gravity (Gs) as high as 4.5. Barite was produced in the United States in 1845 and was used originally as a filling material in production of white paint. In 1926, the consumption and importance of barite increased significantly when it was began to be used in drilling muds . It was reported that in 2017, 70% of barite was consumed as weighing agent for drilling muds activities . It is known that the attenuation coefficient influenced by material density. As for concrete, it's density depends mostly on the density of the aggregates . Therefore, the higher the contents of barite in concrete, the higher the attenuation coefficients . However, barite is brittle material that not suitable to be used as aggregates for structural concrete production . The linear attenuation coefficient () of a 10 mm thick of material (concrete) subjected to radiation emission can be determined by formula proposed by Beer-Lambert law as: I = I0 e-μx (1) Where x is the sample thickness, I and I0 are the number of counts recorded in the detector with and without the shielding targets, respectively. Plotting each ln (I0/I) versus x would give a straight line and μ was obtained using the value of the slope Numerous studies on the use of barite as radiation shielding have been conducted. Akkurt et al stated that barite is one of the most effective materials used as an aggregate in heavy-weight concrete production, and it can be used as radiation shielding effectively. The effect of barite content in geopolymer fly ash concrete has been studied by Muhammed et al. . It was found that the linear attenuation coefficient of the regular concrete increases with the higher the percentage of barite in the concrete. However, the linear attenuation coefficient of geopolymer fly ash concrete was lower than that of normal concrete Zorla et al investigated the effectiveness of high strength concrete mixed with basalt fibers, which was infused with boron, to protect emission against gammaray radiation. It was stated that the mix could absorb radiation emission better than that of regular normal concrete, so that it can be used to reduce the thickness of the concrete wall as radiation protection. Chen developed precast board consists of fiber concrete layers and high density concrete barite purposedly used as radiation shielding. It was reported that the board was effective to protect radiation emission and easy to be installed in medical hospitals. Akarslana et al. studied the absorption capability of barite coated fabrics subjected to radiation. It was mentioned that linear attenuation coefficient of barite coated fabrics increases up to 28%.
Objectives The purpose of this study is to investigate the effect of barite content to the attenuation coefficient of concretes subjected to gamma-rays radiation. Two grades of concrete samples, namely fc'25 MPa and fc'35 MPa, were tested using CS-137 source of energy, which produced gamma-rays of 662 kV or exposure of 46.427 mGy.
Testing program Two grades of concrete specimen having compressive strength of fc'25 MPa and fc'35 MPa were formulated. For each concrete grade, three set of different types of samples were prepared. The first set of samples was normal concrete using natural sand and crushed stone as aggregates. The second set of samples was concrete with natural sand as fine aggregate and barite used as coarse aggregate. While the last set of samples was concrete incorporated barite as both fine and coarse aggregates. The thickness of the samples was 3 cm, 6 cm, and 10 cm. The summary of the tested samples is presented in Table 1 .
Table 1. The tested samples
No
Aggregates The samples were prepared using Ordinary Portland Cement (Type 1). Cement content of the concrete having compressive strength of fc' 25 MPa and fc' 35MPa was 320 kg/m 3 and 415 kg/m 3 , respectively. Water cement ratio of the specimen was 0.61 for fc'25 MPa and 0.47 for fc'35 MPa. While the gradation of the fine and coarse aggregates both natural and barite are presented in Fig. 1 and Fig. 2 , respectively.
Fig. 1. The gradation of fine aggregates
Fig. 2. The gradation of coarse aggregates Crushing values of crushed stones and barite used in the study was 36% and 43%, respectively. The barite consists of 94.3% of barium sulfate (BaSo4)
Testing procedure The concrete sample was subjected to gamma-rays emitted from the source CS-137 (Fig. 3 ) that emanates energy of 662 kV or exposure of 46.427 mGy. The detector apparatus (Fig. 4 ) to measure the exposure of radiation was located at the distance of 200 cm from the source. The duration of testing was 60 seconds. The exposure of the gamma-rays that passing of each samples was recorded.
Results The recorded exposure and calculated linear attenuation coefficient () of each samples for both concrete grades fc' 25 MPa and fc' 35 MPa were in Table 2 and Table 3 , respectively. The comparison of exposure between specimen fc' 25 MPa and fc' 35 MPa is presented in Figure 5 and Figure 6 , respectively. It can be seen that the linear attenuation coefficient of the samples increases with the percentage of barite content. The average linear attenuation coefficient of the specimen having compressive strength fc' of 25 MPa is 0.173 cm -1 for the samples using natural aggregates, 0.225 cm -1 for the sample using barite as coarse aggregate, and 0.246 cm -1 for the samples using barite as aggregates. While the average of linear attenuation of the specimen fc' 35 MPa is 0.190 cm -1 for the samples using natural aggregates, 0.254 cm -1 for the sample using barite as coarse aggregate, and 0.277 cm -1 for the samples using barite as aggregates, as shown in Table 4 . The relationship between density and linear attenuation coefficient of several materials is presented in Table 5 and Fig. 7 . It shows that the linear attenuation coefficient increases with the density of material. Akyuz stated that the linear attenuation coefficient can be correlated to the material density as: = 0.006 e 1.04 cm -1 ( 2 ) Where is density in kg/dm 3 Using the available data, the linear attenuation coefficient of material subjected to gamma-ray radiation emission might be approximated linearly as: = 0.0001 -0.0855 cm -1 (3) Where: = linear attenuation coefficient (cm -1 ) = density in kg/m 3 Adopting the form of formula proposed by Akyuz , correlation between the linear attenuation coefficient and material density can be predicted using the expression: = 0.047 e 0.00054 cm -1 (4) Where is density in kg/dm 3 The correlation between linear attenuation coefficients and material densities of available data and approximation expressions (Eqn.2 and Eqn.4) are presented in Fig. 8 , 0
Conclusion Density of barite concrete was 32% to 54% heavier than that of normal concrete. There was only concrete specimen with barite as both fine and coarse aggregates that exhibit density larger than 3200 kg/m 3 , which can be used as radiation shielding against gamma-rays Linear attenuation coefficient of the specimen increases with the increase of barite content in concrete. Linear attenuation coefficient of the specimen increases with the increase of the concrete strength. Authors acknowledge the assistance from "Balai Pengamanan Fasilitas Kesehatan Surabaya-Indonesia" for providing Cs-137 source of radiation and wish to thank to PT. UNIChem Candi-Sidoarjo-Indonesia for providing barite material. Fig. 3 . 3 Fig. 3. Setup of the tested sample
Fig. 4 . 4 Fig. 4. Gamma-ray exposure detector apparatus
Fig. 7 .Fig. 8 . 78 Fig. 7. Relationship between linear attenuation coefficient and density
Table 2 . 2 Linear attenuation coefficients of the specimen of fc' 25 MPa Linear Specimen Thickness (cm) Exposure (mGy) attenuation coef. (cm -1 ) 3 25.126 0.205 Normal concrete 6 19.923 0.150 10 8.842 0.166 Concrete with 3 23.253 0.230 barite as coarse 6 12.356 0.221 aggregate 10 5.018 0.222 Concrete with 3 19.240 0.294 barite as coarse 6 13.573 0.205 and fine 10 4.214 0.240 aggregates
Table 3 . 3 Linear attenuation coefficients of the specimen of fc' 35 MPa Linear Specimen Thickness (cm) Exposure (mGy) attenuation coef. (cm -1 ) 3 23.646 0.225 Normal concrete 6 16.506 0.172 10 8.159 0.174 Concrete with 3 20.597 0.271 barite as coarse 6 9.419 0.266 aggregate 10 4.867 0.226 Concrete with 3 18.668 0.304 barite as coarse 6 9.331 0.267 and fine 10 3.486 0.259 aggregates Fig. 6. Linear attenuation of concrete specimen fc'35
Table 4 . 4 The average linear attenuation coefficients of the specimen fc' 25 MPa and fc' 35 MPa
Specimen Concrete fc' 25 MPa Concrete fc' 35 MPa Density (kg/m 3 ) Linear attenuat ion coef. (cm -1 ) Density (kg/m 3 ) Linear attenuat ion coef. (cm -1 ) Normal concrete 2252 0.173 2323 0.190 Concrete with barite as coarse 3004 0.225 3064 0.254 aggregate Concrete with barite as coarse and 3461 0.246 3464 0.277 fine aggregates
Table 5 . 5 The relationship between density and linear attenuation coefficient MATEC Web of Conferences
The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).
|
10.17344/acsi.2023.8155
|
cc-by
| null | null |
openalex
|
Novel anthraquinone-based cyclic antioxidants containing amino, thio and oxo groups were synthesized and their antioxidant capacity was determined by using the cerium(IV)-reducing antioxidant capacity (CERAC) assay. 1 H, 13 C NMR, FTIR-spectroscopy and ESI (Electrospray Ionization) mass spectrometry were used for the characterization of anthraquinone derivatives exhibiting CERAC antioxidant capacity. This study is of great importance, because the antioxidant capacity of anthraquinone compounds was analyzed for the first time by the CERAC method and CERAC-Trolox equivalent antioxidant capacity (TEAC) values were higher than that for Trolox. It should also be noted that, since all synthesized anthraquinone derivatives have the potential to find applications in terms of their biological properties due to their sulfur and nitrogen content, they will make an important contribution to the literature.Introductıon Anthraquinones (anthracene-9,10-dione) constitute a very important class of compounds in drug production due to their high biological activities. Antioxidant, antifungal, antiviral, antimicrobial, antidiabetic are among the most interesting pharmacological activities of anthraquinone compounds; thus they have been a focus of a wide range of studies in recent years. In addition to their pharmacological properties, there are many natural and synthetic anthraquinone derivatives that find application for imaging devices, in cosmetics, textile, food and paint industry products. In all these areas of use, separation processes of pollutants with redox potential by various chemical and biochemical processes can also be added. 9, 10 Antioxidants are molecules that delay or prevent damage to the cell structure by inhibiting free radical reactions or by promoting their decomposition. 11 Most heart and cancer diseases occur as a result of low antioxidant limit. 12, 13 Antioxidants protect against many genetic disorders such as asthma, cancer, eczema, aging and cataracts. 14 Nitrogen-and sulfur-containing 9,10-anthraquinone derivatives are widely used in chemistry and technology and exhibit a wide range of biological properties such as anticancer, antimicrobial and antioxidant. Nucleophilic substitution reactions of dichloroanthraquinones are a highly preferred type of reaction as they allow their easy functionalization. There are many studies in the literature examining the mono-and/or di-product formation conditions as a result of the selective nucleophilic substitution reaction of 1,5-dichloroanthraquinone with various nucleophiles. Most dichloroanthraquinone compounds have been successfully synthesized via the Buchwald-Hartwig cross-coupling reaction or by the Ullmann coupling reaction. For this type of coupling reaction, whether the nucleophile has terminal electron-donating alkylamino groups is important for reaction efficiency. If terminal electron donor groups are absent, the reaction will take place in higher yield. For example, in a published study, the reactions of alkylphenylamine and alkylphenylsulfonamide without terminal electron-donating alkylamino groups occurred smoothly, yielding the corresponding Bektas and Onul: Synthesis and Determination of Cerium(IV)-Reducing ... arylamino derivatives and arylsulfonamide derivatives in good yields (62-98%) (Scheme 1). 20 The nucleophiles used can also allow the formation of the disubstituted anthraquinones along with the mono product in the reaction. For example, in the same study, nucleophiles containing a terminal tert-butyl group were used to increase solubility and to carry out the reaction to form 1,5-disubstituted anthraquinone derivatives. In the reaction with N,N-dimethylaminophenylsulfonamide, 1,4-and 1,8-disubstituted anthraquinone derivatives were obtained in reasonable yield, while 1,5-disubstituted product could not be obtained under the same reaction conditions. Considering that the electron donating property of the terminal NH 2 group, which is suitable for the Buchwald-Hartwig cross-coupling reaction, is reduced due to the electron accepting neighboring sulfone unit, it is concluded that this limited reactivity is due to the limited solubility of these derivatives (Scheme 2). 20, 26 Anthraquinone derivatives that can be used as antioxidants are very good electron and hydrogen donors. However, their radical structure is relatively stable due to their resonance delocalization. Interpretation of antioxidant values of anthraquinones is quite difficult due to the existence of different types of molecular radical scavenging mechanisms and their antioxidant properties depending on the structure. 27 Although the antioxidant activity determinations of anthraquinones have been studied with most of the classical methods known before, as far as we know, they have never been investigated with the CERAC assay (Table 1 ). 28, 29 The basis of the CERAC method is based on electron transfer between Ce(IV) and antioxidant compound in an acidic sulfate-containing medium (i.e., H 2 SO 4 and Na 2 SO 4 ). In this redox reaction, antioxidant compounds are oxidized and Ce(IV) ions are reduced to Ce(III) ions. Meanwhile, the absorbance of Ce(IV) decreases after interacting with the antioxidant molecule. 36 The reasons for Bektas and Onul: Synthesis and Determination of Cerium(IV)-Reducing ... using sulphate media in this method are: (i) Ce(IV)/Ce(I-II) reduction potential is low when sulphate ions are sufficiently present; (ii) the ability of Ce(IV) to complex with organic molecules is reduced; and (iii) Ce(IV) sulphate is of long-term stability in the solutions in the environment containing H 2 SO 4 .
Experimental
1. Chemicals and Apparatus Unless specifically indicated, all chemicals and reagents used in this study were purchased from commercial sources and used without purification. Chemicals used for synthesis and CERAC analysis of anthraquinone derivatives were: 1,5-dichloroanthraquinone, allylamine, 3,3'-diaminodiphenylsulfone, para-aminobenzenesulfonamide, 2-(ethylmercapto)ethanol, ethylene glycol, Trolox (Sigma-Aldrich), Ce(SO 4 ) 2 4H 2 O and H 2 SO 4 (Merck). FTIR, ESI-MS, 1 H and 13 C NMR spectra were recorded with Shimadzu IR Spirit Spectrophotometer, Shimadzu LC-MS 8045 Triple Quadrupole Mass Spectrometer and Agilent VNMRS 500 MHz Nuclear Magnetic Resonance Spectrophotometer, respectively. Melting points were recorded by digital melting point equipment Büchi SMP20 (B-540). CERAC antioxidant assay studies were carried out at Istanbul University Plant and Herbal Products Application and Research Center.
1-Synthesis of Anthraquinone Derivatives 3a-d Anthraquinone derivatives were synthesized by a similar method to that in the literature (Scheme 3). 40 Based on the application of this method, after adding 25 mL of ethylene glycol to the 3.6 mmole of 1,5-dichloroanthraquinone compound, nucleophile and potassium hydroxide at an equimolar ratio were added to the mixture. Thereafter the mixture was refluxed at 110-120 C for 48 h, the product was obtained by column chromatography, if necessary, and dried in a vacuum oven at 40 C.
3. 2-Characterization of Synthesized Compounds
4-((5-Chloro-9,10-dioxo-9,10-dihydroanthracen-1-yl) amino)benzenesulfonamide (3c). Obtained by the reaction of 1 (1 g, 3.6 mmol) and para-aminobenzenesulfonamide (2c) (0.62 g, 3.6 mmol). Product is yellow oily substance, 0.175 g (11.7%), R f 0.60 (ethyl acetate). FTIR (ATR, cm -1 ): 3400, 3300 (-NH), 3091 (-CH aromatic ), 1640, 1580 (C=O), Reducing power may be attributed to hydrogen donating ability. 35 Bektas and Onul: Synthesis and Determination of Cerium(IV)-Reducing ...
(S=O) .
1-((3-((3-Aminophenyl)sulfonyl)phenyl)amino)-5chloroanthracene-9,10-dione (3d). Obtained by the reaction of 1 (1 g, 3.6 mmol) and 3,3'-diaminodiphenylsulfone (2d) (0.89 g, 3.6 mmol). Product is yellow solid, 0.136 g (7.7%), R f 0.56 (ethyl acetate), m.p. 173-174 C. FTIR (ATR, cm -1 ): 3360, 3290 (-NH), 3100 (-CH aromatic ), 1740, 1680 (C=O), 1295 (S=O). 1
4. 3-CERAC (Cerium(IV) Reduction Antioxidant Capacity) Method The Trolox equivalent antioxidant capacity of an antioxidant molecule is the millimolar (mM) concentration of the Trolox solution, which has the same reducing power as 1 mM antioxidant solution under the same conditions. Furthermore, the measure of total antioxidant capacity (TAC) refers to the cumulative effect of all antioxidants present in the compound. 36, 41 In the laboratory study to determine the CER-AC antioxidant activities of anthraquinone compounds, Ce(SO 4 ) 2 solution was added to anthraquinone solution prepared in DMSO at certain concentrations in an acidic sulfate-containing medium. After the reaction mixture was left for 30 min at room temperature, absorbance measurements were made at 320 nm, which is the maximum absorption wavelength of Ce(IV). Since the initial absorption of Ce(IV) decreases after interacting with antioxidant molecules, the ratio of the antioxidant molar absorptivity coefficient obtained from the measured absorbance values to the molar absorptivity coefficient obtained from the concentration-absorbance plot of Trolox gives the TEAC (Trolox equivalent antioxidant capacity) value.
Results and Discussion In this study, we prepared systematically a series of arylaminoanthraquinone derivatives containing electron accepting sulfone units and electron donating dialkylami- no or oxo units, by refluxing 1,5-dichloroanthraquinone compound with amino, thio and oxo nucleophile in basic KOH medium in ethylene glycol at 110-120 C. This synthesis method, besides being economical and practical, is a one-step (one pot synthesis) reaction and makes it possible to obtain pure products without the need for hard-to-find and expensive catalysts. 40 Reaction between nucleophiles and 1,5-dichloroanthraquinones is driven by the S N Ar mechanism (aromatic nucleophilic substitution reaction). The hydrogen bond between the ethylene glycol and the oxygen of the carbonyl group increases the electrophilicity of the anthraquinone, facilitating the attack of the nucleophile (Scheme 4.) Amino-and sulfur-containing derivatives of 9,10-anthraquinones have received great attention for their beneficial antioxidant properties. 16 In this context, based on this bioactivity, we preferred amino-, sulphoand sulfur-containing nucleophiles, which can significantly affect the antioxidant properties of anthraquinones, in order to develop new and powerful biological agents of synthetic origin. FTIR spectra of compounds 3b-d showed clear absorption bands at 3500-3290 cm -1 , 1740-1580 cm -1 and 1583-1520 cm -1 belonging to υ(NH) amine, υ(C=O) and υ(C=C) aromatic, respectively. Furthermore, υ(C-O) band of 3a and υ(S=O) bands of compound 3c-d appeared at 1055 cm -1 and 1261-1295 cm -1 , respectively. For compounds 3a-d, the aromatic protons of the anthraquinone moiety gave resonance signals in 1 H NMR spectra between 7.90-7.27 ppm as multiplets, doublets and triplets. The -OCH 2 and -SCH 2 protons of compound 3a showed resonance signals at 4.90 and 2.48-2.40 ppm, respectively. The signal of the -NH proton for compound 3b was observed at 4.88 ppm, while it was around 8.10 ppm for both 3c and 3d. In addition, the -NH 2 proton signals for compounds 3c and 3d were observed at about 4.87 ppm. The carbon atom resonance signal of the carbonyl group of anthraquinone derivatives 3a-d in 13 C NMR was observed in the range of 189.0-180.0 ppm. The 1 H, 13 C NMR and FTIR spectroscopic data of the obtained compounds are in agreement with similar anthraquinone derivatives described in the literature. 19, 20, 42 The presence of an electron accepting sulpho unit between amino and aryl units, the terminal group containing an electron donating amino unit, the number and position of nitrogen and sulfur atoms in the structure contributed significantly to the decrease of yield and antioxidant activity. Since mercaptoethanol and allylamine nucleophiles do not contain terminal electron-donating alkyl amino groups, their reactions proceeded more smoothly, producing the corresponding 3a and 3b anthraquinone derivatives in higher yields than 3c and 3d. Because of the fact that, 3,3'-diaminodiphenylsulfone and para-aminobenzenesulfonamide contain strong electron donating alkylamino terminal groups, their reactions with 1,5-dichloroanthraquinone have not progressed completely. On the other hand, during the synthesis of 3b not only the monoproduct (3b), but also the disubstituted anthraquinone derivative was formed with the removal of both chlorines as a result of the nucleophilic substitution reaction. When the synthesis of 3b was terminated, it was observed that the amount of 1,5-dichloroanthraquinone compound still present in the reaction medium was higher than the amount of unreacted anthraquinone in the synthesis of 3c and 3d. In the nucleophilic substitution reactions of 2a and 2c as nucleophiles reacting with 1,5-dichloroanthraquinone, it can be thought that further reactivity of the monoproduct is more suppressed due to the fact that the electron withdrawing properties of the monochloroanthraquinone derivatives (3a and 3c) formed in the reaction are more reduced compared to 3b and 3d by the addition of electron donating nucleophiles to the anthraquinone. 20 TEAC and TAC values of compound 1 and its syn- It is difficult to interpret the antioxidant activity properties of anthraquinone compounds. Because the antioxidant effect, the radical capture molecule mechanism, may differ in terms of structure-dependent characteristics and environmental effects. 43 Anthraquinones are good electron and proton donors. In addition, radical structures show relative stability due to the resonance delocalization. 44 In the CERAC method, electron transfer takes place. 36 According to the results of the CERAC analysis, starting 1,5-anthraquinone compound (1) did not exhibit antioxidant activity unlike its derivatives; this can be explained by the fact that it does not contain nitrogen and sulfur atoms in its structure. Among the analyzed samples, compound 3c showed the highest TEAC and TAC values determined by CER-AC assay. It can be interpreted that it originates from the sulpho (O=S=O) group and -N atom in its structure. In addition, the increased stability as a result of the increase in electron density on the carbon atom to which the sulpho group is attached, through delocalization caused by the unshared electron pair in the -NH atom in this compound, may have affected the activity. Considering the free structure effects of atoms on the antioxidant activity, it can be commented that the low number of bonds of the sulfur atom in compound 3a may have contributed to high antioxidant activity compared to 3d.
Conclusıons A simple metal-free alternative procedure for Ullmann type C-N coupling reactions has been carried out by allowing dichloroanthraquinones to react with a variety of nucleophiles in the presence of ethylene glycol. In conclusion, we have successfully developed a simple, moderate, economical and environmentally friendly procedure for the synthesis of N-and O-aryl anthraquinone derivatives under metal-free conditions. 40 The fact that the derivatives of 1,5-anthraquinone containing similar groups to those introduced by our nucleophiles have not been studied much, increases the importance of the study. 20, 45, 46 In addition to what we said, the reason why the CER-AC method was preferred for the determination of antioxidant activity is that it is easy, does not require a very high level of equipment, and as far as we know, the antioxidant activities of anthraquinones have never been studied with this method before. According to the results of the analysis, while the 1,5-dichloroanthraquinone starting compound (1) did not exhibit CERAC antioxidant activity, all the synthesized derivatives responded to the CERAC method. Scheme 1 . 1 Scheme 1. Nucleophilic substitution reactions of dichloroantraquinones.
13 C NMR (126 MHz, DMSO-d 6 ): δ (ppm) 188.0, 180.0, 160.0, 161.0, 138.0, 136.0, 133.0, 122.0. ESI+: m/z 298.3 [M+H] + , C 17 H 12 ClNO 2 (M A = 297.7 g/mol).
1 H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 8.10 (bs, 1H, -NH), 7.85-7.75 (m, 4H, -CH aromatic ), 7.63-7.58 (m, 4H, -CH aromatic ), 7.29 (d, J = 8.0 Hz, 2H, -CH aromatic ), 4.87 (bs, 2H, -NH 2 ). 13 C NMR (126 MHz, DMSO-d 6 ): δ (ppm) 189.0, 181.0, 160.0, 136.0, 120.0, 118.0. ESI: m/z 414.0 [M+H] + , C 20 H 13 ClN 2 O 4 S (M A = 412.8 g/mol).
H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 8.10 (bs, H, -NH), 7.80-7.73 (m, 4H, -CH aromatic ), 7.61-7.56 (m, 5H, -CH aromatic ), 7.27 (d, J = 8.30 Hz, 5H, -CH aromatic ), 4.89 (bs, 2H, -NH 2 ). 13 C NMR (126 MHz, DMSO-d 6 ): δ (ppm) 188.0, 181.0, 162.0, 160.0, 138.0, 136.0, 124.0, 116.0. ESI+: m/z 489.9 [M+H] + , C 26 H 17 Cl-N 2 O 4 S (M A = 488.9 g/mol).
Scheme 3 . 3 Scheme 3. Synthesized anthraquinone compounds.
Scheme 4 . 4 Scheme 4. Possible mechanism of nucleophilic substitution reaction.
(Allylamino)-5-chloroanthracene-9,10-dione (3b). Obtained by the reaction of 1 (1 g, 3.6 mmol) and ally- lamine (2b) (0.21 g, 3.6 mmol). Product is orange solid, 0.196 g (18.32%), R f 0.64 (ethyl acetate), m.p. 155-156 C. FTIR (ATR, cm -1 ): 3502 (-NH), 3080 (-CH aromatic ), 2949 (-CH aliphatic ), 1648, 1585 (C=O). 1 H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 7.83-7.76 (m, 3H, -CH aromatic ), 7.62-7.58 (m, 2H, -CH aromatic ), 7.29 (dd, J 1 = 8.40 Hz, J 2 = 1.1 Hz, H, -CH aromatic ), 4.88 (bs, H, -NH), 4.17 (m, 3H, =CH allylic , =CH 2allylic ), 3.80 (t, J = 4.90 Hz, 2H, -NCH 2 ). 1-Chloro-5-(2-(ethylthio)ethoxy)anthracene-9,10-dione (3a). Obtained by the reaction of 1,5-dichloroanthraqui- none (1) (1 g, 3.6 mmol) and 2-(ethylmercapto)ethanol (2a) (0.38 g, 3.6 mmol). Product is yellow solid, 0.359 g (28.72%), R f 0.54 (ethyl acetate), m.p. 164-165 C. FTIR (ATR, cm -1 ): 3100 (-CH aromatic ), 2933 (-CH aliphatic ), 1650, 1583 (C=O), 1260, 1055 (C-O). 1 H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 7.90-7.74 (m, 3H, -CH aromatic ), 7.60 (t, J = 7.50 Hz, 2H, -CH aromatic ), 7.27 (d, J = 8.0 Hz, H, -CH aromatic ), 4.90 (t, J = 5.40 Hz, 2H, -OCH 2 ), 2.48-2.40 (m, 4H, -SCH 2 ), 1.30-1.26 (m, 3H, -CH 3 ). 13 C NMR (126 MHz, DMSO-d 6 ): δ (ppm) 189.0, 182.0, 138.0, 136.0, 122.0, 118.0. ESI+: m/z 364.5 [M+NH 4 ] + , C 18 H 15 ClO 3 S (M A = 346.8 g/mol). 1-
Table 1 . 1 Antioxidant activities and mechanisms of some anthraquinones Anthraquinone Experimental Method Mechanism Purpurin DPPH (1,1-diphenyl-2-picrylhydrazyl) radical Can be associated with 3 hydroxy scavenging capacities. groups. 30 Alizarin Trolox (6-Hydroxy-2,5,7,8-tetramethylchro- Can be associated with electron or man-2-carboxylic acid) equivalents (measure the hydrogen atom transfer. 31 free radical scavenging activity). Emodin DPPH radical scavenging capacities. Can be associated with intermolecular Physcion hydrogen bridges and asidic portion's Emodin-8-O-β-D-idopyranoside ability to donate H and form the oxidized structure. 32 Asphodeline anatolica Radical scavenging activities measured using Antioxidant molecules interact with Asphodeline baytopae DPPH radical. DPPH and ABTS radicals via electron Asphodeline brevicaulis Phosphomolybdenum and β-carotene bleaching or hydrogen atom transfer, converting Asphodeline cilicica methods. them to a stable structure or a ABTS (2,2'-azino-bis(3-ethylbenzthiazoli- non-radical species. ABTS activity can ne-6-sulfonic acid) radical cation were expressed be explained as dependent on as trolox equivalents. flavonoid level. The reducing power measured using cupric ion High reducing power may be attribut- reducing activity (CUPRAC). ed to high polyphenol and flavonoid Ferric ion reducing antioxidant power (FRAP). content of structure. 33 Metal chelating activity on ferrous ions was expressed as EDTA equivalents. Alaternoside DPPH radical scavenging capacity. May be attributed to Physcion-8-O-rutinoside the higher number of hydroxyl groups. 34 Rhamnocitrin Aloe AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) were generated peroxyl radicals scavenging capacity. CUPRAC
Scheme 2. Synthesis of disubstitue anthraquinones.
Bektas and Onul: Synthesis and Determination of Cerium(IV)-Reducing ...
|
10.21203/rs.2.19552/v3
|
cc-by
| null | null |
openalex
|
Objective: To investigate the relationship between Modic changes (MCs) and recurrent lumbar disc herniation (rLDH), and that between the herniated disc component and rLDH following percutaneous endoscopic lumbar discectomy (PELD). Methods: We included 102 (65 males, 37 females, aged 20-66 yrs) inpatients who underwent PELD from August 2013 to August 2016. All patients underwent CT and MRI preoperatively. The presence and type of Modic changes were assessed. During surgery the herniated disc component of each patient was classi ed into two groups: nucleus pulposus group, hyaline cartilage group. The association of herniated disc component with Modic changes was investigated. The incidence of rLDH was assessed based on more than 2-year follow-up. Results: In total, 11 patients were lost to follow up; the other 91 were followed up during 24-60 months. Of the 91 patients, 99 discs underwent PELD; 28/99 (28.3%) had MCs. Type I and II MCs were seen in 9 (9.1%) and 19 (19.2%), respectively; no type III MCs were found. Among 28 endplates with MCs, according to the herniated disc component, 18/28 (64.3%) showed evidence of hyaline cartilage in the intraoperative specimens, including 6/9 and 12/19 endplates with type I and II MCs, respectively. Among 71 endplates without MCs, 14/71 (19.7%) showed evidence of hyaline cartilage in the intraoperative specimens. Hyaline cartilage was more common in patients with MCs (P<0.05). We found 2 cases of rLDH in the non-MC group (n=71); 6 cases rLDH were found in the MC group (n=28), including 2 and 4 cases for types I and II, respectively. There was no signi cant difference between types I and II (P>0.05). rLDH was more common in patients with MCs (P<0.05). We found 5 rLDH cases in the hyaline cartilage group (n=32); 3 rLDH cases were found in the nucleus pulposus group (n=67). rLDH was more common in the hyaline cartilage group (P<0.05). Conclusions: rLDH following PELD preferentially occurs when MCs or herniated cartilage are present.Introduction Percutaneous endoscopic lumbar discectomy (PELD), a minimally invasive spinal procedure, has become increasingly well accepted by both surgeons and patients who suffer from lumbar disc herniation (LDH). Compared with conventional open surgery, this technique allows the removal of the herniated disc through a very small skin incision. However, along with the widespread use of PELD, many researchers have stated concerns regarding recurrence. [2, 3] Recurrent lumbar disc herniation (rLDH) has been de ned as disc herniation at the same level with the reappearance of the same pain after a pain-free interval and magnetic resonance imaging (MRI) con rmation. The recurrence rate of LDH has been reported to be 5-15%. There have been many studies designed to determine the recurrence of LDH, and various risk factors were suggested including disc degeneration, trauma, age, smoking, gender, and obesity. [3, 4] Radiologically identi able factors, such as disc degeneration, disc height, and sagittal range of motion have been shown to be related to spinal instability and consequently to rLDH. Modic changes, which are present as signal alterations in the vertebral endplate and adjacent bone marrow, are found on T1-and T2-weighted MRI. These changes are associated with vertebral endplate ssures and disc herniation. . However, little is known about the relationship between MCs and rLDH. MCs are known to be associated with LDH-containing cartilaginous fragments. Schmid et al. reported the presence of a cartilaginous endplate in the extruded disc material in 63% of patients with MCs and the association between the cartilage endplate and spontaneous resorption has been speci cally investigated. However, the relationship between LDH-containing cartilaginous fragments and rLDH has received little attention. The purpose of this study was to investigate the relationship between MCs and rLDH, and that between the herniated disc component and rLDH following PELD.
Materials And Methods
Inclusion criteria Patients were included if they had radicular pain for at least 3 months that was refractory to 6 weeks of conservative treatment with or without neurological de cit, numbness in the lumbar spine, buttock, and/or lower extremity; and MRI and computed tomography (CT) demonstrated anatomical LDH correlating with symptoms and no disc calci cation.
Exclusion criteria Patients were excluded if they had any of the following: prior lumbar surgery at another institution, disc calci cation, segmental instability, vertebral fractures, spinal infection, other types of degenerative disc disease, tumors, or pregnancy.
Recurrent disc herniation Recurrent herniation was de ned as follows: (1) patients showed a successful PELD operation as con rmed by a pain-free interval of at least one month and (2) reappearance of the same pain as presentation and MRI con rmation of the recurrent herniation on the same level.
CT and MRI CT scan of the lumbar spine was performed using a 16-slice CT scanner (GE LightSpeed Pro 16; GE Healthcare) with a detector con guration of 16×1.25 mm. A standard lumbar spine protocol with a tube voltage of 120 kV, tube current of 100-650 mA, and a rotation time of 0.8 seconds was used. Automatic tube current modulation based on the patient's size and X-ray attenuation was used. The slice thickness and reconstruction interval were 1.25 mm and 0.625 mm, respectively. MRI images of the lumbar spine were obtained using a General Electric 1.5-T magnet with a T1-weighted sequence (repetition time/echo time, 560 ms/12 ms; eld of view, 320×256; receiver bandwidth, variable; 4.0-mm slice with a gap of 1.0 mm; number of excitations, 3) and a T2-weighted sequence (repetition time/echo time, 3000 ms/100 ms; eld of view, 320×256; receiver bandwidth, variable; 4.0-mm slice with a gap of 1.0 mm; number of excitations, 3).
Patients According to the inclusion and exclusion criteria, we included the inpatients who underwent PELD from August 2013 to August 2016, the patients were consecutively enrolled. All patients had undergone CT and MRI examinations that were evaluated retrospectively. CT was used to evaluate whether the herniated disc component had a calci cation. The presence, location, and type of MCs were assessed from the MRI scans. According to the herniated disc component in the intraoperative specimens, the herniated disc component of each patient was classi ed into two groups: the nucleus pulposus group and the hyaline cartilage group. The association of herniated disc component with MCs was investigated. Moreover, patients were also divided into the MCs group and non-MCs group. The incidence of rLDH was compared between the nucleus pulposus and hyaline cartilage groups, and between the MCs and non-MC groups during a follow-up period of at least 2 years (Figure 1 ). Ethical approval was obtained from the Medical Ethics Committee of the hospital. Additionally, all patients provided written informed consent for their information to be stored in the hospital's database and used for research.
Data analysis Data were inputted into Excel (Microsoft Corp., Redmond, Washington) and quantitative results were expressed in terms of a mean and standard deviation. Data were transferred to SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Group t-tests (two-sided), and chi-square tests, were used to compare mean values, and proportions, respectively. A signi cance level of 0.05 was used, and p<0.05 was considered signi cant without multiple test adjustment.
Results In total, 102 patients(65 males, 37 females; age, 50.9±7.9 years; age range, 20-66 years) underwent PELD and were enrolled in the study, 11 patients were lost to follow up; the other 91 patients (57 males, 34 females, age, 50.8±7.8 years; age range, 20-66 years) were followed up for more than two years (24-60 months). Of the remaining 91 patients, 99 discs underwent PELD, and 28/99 (28.3%) had MCs; type I and II MCs were seen in 9 (9.1%), 19 (19.2%), respectively. No type III MCs was found. MCs and hyaline cartilage Among 28 endplates with MCs, according to the herniated disc component, 18/28 (64.3%) showed evidence of hyaline cartilage in the intraoperative specimens, including 6/9 endplates with type I MCs and 12/19 endplates with type II. There was no signi cant difference between types I and II (P>0.05). Among 71 endplates without MCs, 14/71 (19.7%) showed evidence of hyaline cartilage in the intraoperative specimens. Hyaline cartilage was more common in patients with MCs (P<0.05).
MCs and rLDH rLDH was more common in patients with MCs (P<0.05)(Table 1 ). There were 2/71(2.8%) cases of rLDH in the non-MC group (n=71); 6/28(21.4%) cases of rLDH were found in the MC group (n=28), including 2 and 4 cases of types I and II, respectively. There was no signi cant difference between types I and II observations (P>0.05).
Hyaline cartilage and rLDH was more common in the hyaline cartilage group (P<0.05) (Table 1 ). There were 5/32(15.6%) cases of rLDH in the hyaline cartilage group (n=32) and 3/67(4.5%) cases of rLDH in the nucleus pulposus group (n=67).
Discussion
Summary of ndings To our knowledge, this study is the rst to investigate the relationship between MCs and rLDH, and that between the herniated disc component and rLDH following PELD. We found that hyaline cartilage and rLDH were more common in patients with MCs. rLDH was more common in the hyaline cartilage group. rLDH following PELD preferentially occurred when MCs or the herniated cartilage are present.
MCs and herniated cartilage The variable composition herniated tissue has been described previously, with various proportions of nucleus pulposus, annulus, hyaline cartilage, and bone being reported. Tanaka et al. suggested that the cartilaginous endplate-osteochondral junction is weak, whereas the cartilaginous endplate-inner annulus brosus connection is strong, so herniated tissue usually contains a cartilaginous endplate. Rajasekaran et al. reported that a high proportion of Indian patients had herniations containing cartilage and bone. MCs, which present as signal alterations in the vertebral endplate and adjacent bone marrow, are found on T1-and T2-weighted MRI. MCs are known to be associated with LDH-containing cartilaginous fragments. Schmid et al. reported the presence of a cartilaginous endplate in the extruded disc material in 63% of patients with MCs. Our study con rmed the results; 64.3% of our patients with MCs showed evidence of hyaline cartilage in the intraoperative specimens, only 19.7% showed evidence of hyaline cartilage. Hyaline cartilage was more common in patients with MCs.
MCs and rLDH Our study showed that rLDH preferentially occurred when MCs or the herniated cartilage were present. For patients with MCs, these conditions usually imply that the endplate structure has been damaged. The herniated cartilaginous endplate is only one part of the damaged endplate, because the connection between the cartilage endplate and the vertebral body is relatively weak, so other parts of the endplate easily separate from the vertebral body and herniate with the nucleus pulposus, and rLDH occurs. Similar to the MCs endplate, if the herniated cartilage is present in the intraoperative specimen, which also re ects damage to the endplate structure, other parts of the endplate easily separate from the vertebral body and herniate with nucleus pulposus, and rLDH preferentially occurs. Our study has some limitations. Firstly, it was a retrospective study with a small sample size. The relatively small sample size limits the accuracy of correlation between the rate of rLDH and the type of MCs. Secondly, the study focused on the phenotypic association between rLDH and MCs or the herniated disc component, while no correlative mechanism was studied. In particular, we did not con rm the results using biomechanics and histomorphologic methods. Further study is needed in the future. Nevertheless, this study suggests that rLDH preferentially occurs after PELD when MCs are present. If we choose PELD for patients with MCs, we need to inform these patients that they have a higher incidence risk of rLHD; especially for old patients. Meanwhile, if cartilage is present in the herniated disc component, we also should inform these patients that the they have a higher incidence risk of rLHD. Such patients may require a second operation.
Conclusions rLHD following PELD preferentially occurred when MCs or the herniated cartilage were present. Patients with MCs had a higher incidence risk of rLHD. Figures Figures
Figure 1 Example 1 Figure 1
Table 1 . 1 rLDH Patients data of the MCs and no-MCs group, the hyaline cartilage and nucleus pulposus group rLDH No rLDH p-value MCs group 6 22 0.006 Non-MCs group 2 69 Hyaline cartilage group 5 27 0.029 Nucleus pulposus group 3 64
|
10.1186/1471-2202-14-s1-p18
|
cc-by
| null | null |
openalex
|
A combined power spectral and time-series bifurcation analysis of a neural mass model is presented. Such 'multimodal' analytical techniques are being used in several researches to understand Electroencephalograph (EEG) anomalies in brain disorders , in contrast to 'power spectra-only' analytical studies that were more common during the early days of EEG analysis. In a recent work, a combined analysis of a simple thalamo-cortical neural mass model in context to EEG abnormality in Alzheimer's disease (AD) is presented . The study shows that 'unimodal' analytical techniques such as power spectra-only studies without a simultaneous observation of the timeseries model output may lead to anomalous conclusions and hypotheses. Towards this, in this work, a 'multimodal' analytical technique is applied on a thalamocorticothalamic (tct) model, which was earlier studied usingpower-spectra analysis only . The tct model is an enhanced version of that used in and is based on biological data available in current literature. Furthermore, it aims to mimic thalalmocortical oscillations such as observed in the EEG of both healthy and diseased brain. Here, the power spectra of the tct model output is observed within the δ (1-3 Hz), θ (4-7 Hz), α (8-13 Hz), β (14-30 Hz) bands, along with a simultaneous analysis of the time series behaviour, the latter showing three behavioural modes: noisy point-attractor, spindle and limitcycle. With all parameters at their basal values, the output time series is in a noisy point-attractor mode with maximum power within the alpha band (Figure 1 ). However the model shifts into a limit cycle oscillatory mode with a decrease in inhibitory connectivity parameters in the model (Figure 1 ); the corresponding power spectra show an increase in peak power within the θ and δ bands along with a simultaneous decrease in power within the α and β bands. The model behaviour is very much in agreement with in-vitro studies which report an increased theta band power and a simultaneous decreased alpha band power during transition from wakefulness to sleep. Furthermore, the in-vitro time-series are qualitatively very similar to those obtained using the model. Thus, the model indicates a decreased inhibitory activity to be the neural correlate of the transitive state between wakefulness and sleep. On the other hand, increased mean firing activity of the extrinsic model inputs pushes the model, first into a spindling mode, and then into a limit cycle mode. In this state, the power within the delta band shows a significant increase compared to those within the other frequency bands. This behaviour is more similar to in-vivo studies of awake-to-sleep transition as reported in . Published: 8 July 2013
|
10.1163/23752122-02301012
|
cc-by
| null | null |
openalex
|
Some obscure connections that may have affected aspects of F.M. Dostoevsky's life and works are explored and their implications delved into, with the intention of offering new insights into the forces and processes that encroached onto the dynamics of the writer's artistic production. First translations into English of the documents are included. A. Foregrounded is a letter from Prince Vladimir Meshchersky of early January 1872 (first published in 2014), addressed to the heir to the throne of Russia, Grand Duke Alexander Alexandrovich Romanov. Enclosed with it was a letter from F.M. Dostoevsky in which he asked the heir for financial assistance. In addition, Dostoevsky's other known letters to the heir are evaluated. These include Dostoevsky's next letter thanking the heir for the "favour" granted datedIntroduction More has been written about Dostoevsky than probably any other writer in Russia, yet there are still aspects pertaining to his life and works of which we have only partial knowledge. Not having absolute certainty about some obscure connections should not preclude us from attempting to explore them. It is proposed to go on a quest to consider some such examples, delve into their implications, offer our observations, and come up with possible new insights into the forces and processes that encroached onto the dynamics of Dostoevsky's artistic production. One's justification is Dostoevsky's own expressed belief that there is no one, who consciously yearns to make a mistake, and that more often than not, those who come to the wrong conclusions sincerely seek after truth (as reported by N.N. Strakhov in the context of their evening conversation in Florence, regarding Dostoevsky opposing the view that 2 x 2 could only equal 4, and defending 2 x 2= 5): "By the very essence of the matter, every thought has its cause and its foundation, every thought, both broad and deep, and petty and narrow, moves according to the same logical laws and, consequently, the grossest error contains elements of truth." ["По самой сущности дела всякая мысль имеет свой повод и свое основание, всякая мысль, как широкая и глубокая, так и мелкая и узкая, движется по одним и тем же логическим законам и, следовательно, самое грубое заблуждение носит в себе элементы истины."]1 Hence an attempt will be made to bring to the fore some significant details relating to Dostoevsky that have been neglected by most commentators hitherto.
A Meshchersky's Letter to the Heir to the Throne of Russia Supporting Dostoevsky's Enclosed Request for Financial Assistance, and Further Correspondence. Dostoevsky's appeal to the heir to the Russian throne, Grand Duke Alexander Alexandrovich (1845-1894), the future Alexander iii, was forwarded by Prince Vladimir Petrovich Meshchersky (1839 Meshchersky ( -1914) ) together with Meshchersky's accompanying letter that introduced Dostoevsky's appeal and briefly explained the latter's predicament. First published in Russia in 2014, Meshchersky's letter is being made available to readers here in its first translation into English.2 It has been presumptively dated by the editor of Meshcherky's correspondence as having been written at the "Beginning of January, 1872", while Dostoevsky's letter that it accompanied has not been found so far and is presumed lost. A reference to it is included in the "List of letters and business papers that have not been preserved and not been found 1869-1874" in the Complete Collected Works of F.M. Dostoevsky, where it is presumptively dated as having been written between the "Autumn of 1871-first half of January, 1872".3 Dostoevsky had returned to St Petersburg with his family on July 8, 1871 after living abroad for over four years from mid-April 1867. He was continuing work on his unfinished novel Demons (also known as The Possessed) (Besy), conceived abroad in 1869-1870 that was being serialised throughout 1871 in the monthly journal Russian Messenger (Russkii Vestnik) in which the first 7 parts had appeared in volumes 1-2, 4, 7, 9-11. He is believed to have met Meshchersky sometime in the autumn of that year through the intermediary of A.N. Maikov. At the time Prince Vladimir Meshchersky was a civil servant in the Ministry of the Interior, as well as a published writer and was just embarking on his career as publisher of the weekly-newspaper The Citizen (Grazhdanin) that was launched in January 1872. He had been a friend of Nikolai Alexandrovich Romanov (1843-1865), the heir apparent (and older brother of the addressee of the letter), and since 1863 Meshchersky had corresponded with him and watched him being groomed to succeed his father, Emperor Alexander ii to the throne of Russia. But "tragically" the heir succumbed to a malady and soon died in April 1865 in Nice from tuberculous meningitis of the spine, a disease that appears to have been accelerated by an accidental fall off his horse. As befitting his position, Nikolai had become engaged to the daughter of the King of Denmark, Princess Dagmar (1847 Dagmar ( -1928) ) on his 21st birthday, 8 (20) September 1864, who was present at his deathbed. The following year the Imperial family arranged the betrothal of Grand Duke Alexander Alexandrovich, now the Tsesarevich, to Princess Dagmar after she had accepted Orthodoxy. Nikolai Alexandrovich's passing was the reason why Meshchersky then re-focused his devotion and affection on his grieving younger brother Alexander, the new heir to the throne, and assumed the self-appointed role of becoming his mentor.4 When Meshchersky's letter accompanying that of Dostoevsky was being planned and dispatched, between late 1871 and early 1872, the close relationship between Meshchersky and the young heir had not yet entirely broken off and almost collapsed, as it did for a period of time shortly after. But Dostoevsky is likely to have believed all the while that Meshchersky's relationship with Alexander Alexandrovich was solid. Meshchersky's letter was found amongst his voluminous correspondence with Alexander Alexandrovich begun in 1863, together with the Tsesarevich's unpublished diaries preserved at the State Archive of the Russian Federation (Gosudarstvennyi Arkhiv Rossiiskoi Federatsii-garf) . Meshchersky wrote to the Tsesarevich Alexander Alexandrovich: "I hereby take the liberty of passing on to you a letter -a cry of desperation from the poverty-stricken and celebrated [F. M.] Dostoevsky, the finest writer of our era in Russia, who is at this grievous moment in his life setting his hopes on your mercy!"[«При сем позволяю себе передать Вам письмо -крик отчаяния бедного и знаменитого [Ф. М.] Достоевского, лучшего писателя нашего времени в России, -уповающего в самую тяжелую минуту своей жизни на Ваше милосердие!»]5 According to Meshchersky's explanation: "He (Dostoevsky -I.Z.) came to see me, handed over this letter, and I have been meticulously and conscientiously collecting affidavits in order to have the right to hand this letter over to you. And here it is!" [«Он ко мне пришел, отдал мне это письмо, я собирал справки обстоятельно и добросовестно, дабы иметь право Вам это письмо отдать. И вот!»] Meshchersky attempts to give Dostoevsky a glowing reference, one that was intended to produce the desired positive response from the Grand Duke: Dostoevsk[y] has ended up in an impossible debt situation because, on the death of his brother, in order to save his memory and honest name from shame, he undertook to pay all his debts, took money from various implacable creditors, who are threatening him with prison, and in this desperate situation he is suffering daily epileptic fits which are bringing him close to death. On the other hand, restoring his peace of mind by assisting him with a loan would mean restoring him to life and, with his life, his talent! [Достоевск[ий] потому впал в неоплатный долг, что по смерти брата его взялся, дабы спасти память и честное имя его от позора, заплатить все долги его, получил деньги от разных неумоли-мых кредиторов, которые грозят ему тюрьмою, и в этом отчаянном положении припадки падучей болезни от забот приходят ежедневно и приближают его к смерти. Напротив, возвратить ему спокойствие, помогши ему займом, значило бы возвратить ему жизнь, а с жизнью -талант!]6 Meshchersky's assistance in helping Dostoevsky procure some funding or a loan that enabled the writer to pay off his creditors at least partially, challenges the impression that Dostoevsky's widow Anna Grigorievna wished to create that it was exclusively the venerable (and socially reputable) K.P. Pobedonostsev (1827 Pobedonostsev ( -1908)) , tutor to the heir, who was instrumental in instigating Dostoevsky's contacts with the Grand Duke. Pobedonostsev did indeed facilitate some of Dostoevsky's communications with the Romanov family, but that was at a later stage and under different circumstances, after Dostoevsky's contact with the Grand Duke had already been established with the help of Meshchersky. Pobedonostsev seems to have waited until Dostoevsky's reputation as a Slavophile-orientated writer loyal to the regime had been demonstrated following the publication of Demons, and proven with the launching of his column Writer's Diary (Dnevnik pisatelia) for 1873. Since Dostoevsky's request for financial assistance or a loan was granted by the heir, it confirms one's impression that Dostoevsky must have felt greatly indebted to him and everything that he stood for, and would have felt even more obliged than before to demonstrate his special devotion and support, whenever his conscience allowed it. He was still in the process of completing the concluding chapters of his novel Demons of which two more parts were to be written that would appear in volumes 11-12 in Russian Messenger in 1872. These would provide an acceptable ending that would cohere with the exclusion of the chapter "At Tikhon's" that was again rejected by the publisher M.N. Katkov. The decision to grant Dostoevsky financial assistance would have been made in consultation with the Grand Duke's advisors, since the funds assigned may have had to come out of the official budget allocated to the heir's household, overseen by the Minister of the Imperial Palace, A.V. Adlerberg. It must have been deemed acceptable, or even "gracious and merciful" to give Dostoevsky, a former member of the Petrashevsky circle and political convict, a grant, since he was a popular and respected writer, who proclaimed he had now reformed; besides, he would have been considered valuable to have as an ally. Dostoevsky, in his turn, may have believed that one way of demonstrating his sense of gratitude to the Grand Duke and also to Meshchersky was to offer at the end of 1872 to take up in January 1873 the editorship of the conservative orientated newspaper The Citizen published by Meshchersky, after the latter's current editor G.K. Gradovsky had decided to leave. Significantly, the first issue of The Citizen under Dostoevsky's editorship on January 1, 1873 opened with a tribute to Alexander Alexandrovich entitled: "Joyous tidings on the recuperation of the Heir to the Throne" ["Радостные известия о выздоровлении Государя Наследника»].7 In later issues other articles and items in columns followed in which some current and past members of the Imperial family, and decisions of the government were presented in a positive light, while certain trends in the bureaucracy, and in social ranks or estates received a more critical assessment. Some subsequent letters from Dostoevsky to the heir, Grand Duke Alexander Alexandrovich have been published earlier and are known. The most frequently cited and significant letter is the one of 10 February 1873 (29(1), 260-261), which, according to Dostoevsky's wife Anna Grigorievna was written after Tsesarevich Alexander Alexandrovich had indicated through Pobedonostsev, his tutor, of his interest in learning of Dostoevsky's own interpretation of his novel Demons (29(1), 499).8 However, there does not exist currently any published 7 Irene Zohrab, 'The Contents of The Citizen during Dostoevsky's Editorship: Uncovering the Authorship of Unsigned Contributions. Dostoevsky's Quest to Reconcile the "Flux of Life" with a Self-Fashioned Utopia' . Part 1," The Dostoevsky Journal: An Independent Review, vol. 5,((2003 Review, vol. 5,(( -2004)) correspondence from Pobedonostsev demonstrating this, although he may have mentioned it to Dostoevsky's at one of Meshchersky's gatherings in the winter of 1872-1873 in connection with the publication of the first book-version of Demons. His earliest letters to Dostoevsky begin on 21 June/3 July 1873,9 and concern exclusively formal editorial matters about articles that Pobedonostsev was contributing to The Citizen, while Dostoevsky was its editor, all published anonymously signed with cryptonyms, many relating to his travels in England in early summer and he did not return until the last week of July (29(10, 284) .10 Dostoevsky's decision, while explaining the novel to the Grand Duke was to focus on certain elements in Russian social history that he believed had generated the phenomenon of Nechaevism and attempt to justify its emergence in terms of socio-historical processes, describing his work as a "historical study" [исторический этюд]. In Demons Dostoevsky had depicted provocationally the nihilist ideology espoused by the student Nechaev germinating within Tsarist autocracy. A conspiratorial revolutionary with a pathological tendency, Nechaev's criminal activities were linked by Dostoevsky with the "Westernism" of the liberals of the 1840s, the alleged "fathers" of the nihilists of the 1860s. Now he found himself in the paradoxical situation of having to defend his depiction and justify it to his "august addressee" in a way that would cover all contingencies and possible pitfalls, and simultaneous make his choice of subject matter, that exposed destructive forces in Russian society, legitimate. He advocated a solution by aligning it to an imagined Imperial policy that would accentuate Russian exceptionalism. It was as if he was rearranging the pieces in his kaleidoscopic novel and letting them fall into new patterns that would reshape the outcome accordingly by deflecting blame from Imperial policies and shifting it on to 'historical processes' . (29(1) 260-261): These phenomena are a direct consequence of the age-old divorce of all Russian enlightenment from the native and distinct principles of Russian life. Even the most talented representatives of our pseudo-European development have long since come to be convinced of the absolute crim- inality of us, Russians, dreaming of our distinctiveness. Most horrible of all is that they are absolutely right, because having proudly called ourselves Europeans, we have thereby renounced being Russians. In embarrassment and awe before the fact that we have fallen so far behind Europe in intellectual and scientific development, we have forgotten that we ourselves, in the depth and seeds of the Russian soul, contain within ourselves, as Russians, the ability, perhaps, to bring new light to the world, on condition of the distinctiveness of our development. In ecstasy of our abasement, we have forgotten the most immutable historical law that consists of the fact that without such arrogance about our own significance for the world as a nation we can never be a great nation and leave after ourselves at least something distinctive for the benefit of mankind.11 [Эти явления -прямое последствие вековой оторванности всего просвещения русского от родных и самобытных начал русской жизни. Даже самые талантливые представители нашего псевдоевропейского развития давным-давно уже пришли к убеждению о совершенной преступности для нас, русских, мечтать о своей самобытности. Всего ужаснее то, что они совершенно правы; ибо, раз с гордостию назвав себя европейцами, мы тем самым отреклись быть русскими. В смущении и страхе перед тем, что мы так далеко отстали от Европы в умственном и научном развитии, мы забыли, что сами, в глубине и задачах русского духа, заключаем в себе, как русские, способность, может быть, принести новый свет миру, при условии самобытности нашего развития (29(1) 260-261).] A later note of Dostoevsky's to the Grand Duke Alexander Alexandrovich dated 16 November 1876 was indeed sent at the instigation of Pobedonostsev, who suggested that Dostoevsky present the heir with copies of the Writer's Diary: "I don't know if you are delivering your "Writer's Diary" to the heir, the Tsesarevich? If not, then it wouldn't be bad if you sent it to him ... but if you want to deliver it with an interpretation, then deign to send them to me and I will send it to him with a written explanation that this is from you." [«Не знаю, доставляете ли вы свой "Дневник писателя" наследнику цесаревичу? Если нет, то не дурно было бы, когда б вы ему посылали ... а если желаете доставить с толкованием, то благоволите прислать ко мне и я отошлю их к нему с письменным объяснением, что это от вас представляется»].12 Dostoevsky again assumed the role of an instructor (or 'life coach' in today's parlance) taking the opportunity to outline briefly his view of the Russian historical situation and throw in the popular Slavophile concept of the so-called "Russian Idea": The present great energies in Russian history have elevated the spirits and hearts of the Russian people with unimaginable power to a height of understanding of much that was not earlier understood, and have illuminated in our consciousness the sanctity of the Russian idea more vividly than ever before. I could not fail to respond either, with all my heart to everything that has begun and appeared in our land, in our just and wonderful people. [Нынешние великие силы в истории русской подняли дух и сердце русских людей с недостижимою силой на высоту понимания многого, чего не понимали прежде, и осветили в сознании нашем святыни русской идеи ярче, чем когда бы то ни было до сих пор. Немог и я не отозваться всем сердцем моим на все, что началось и явилось в земле нашей, в справедливом и прекрасном народе нашем. В "Дневнике" моем есть несколько слов, горячо и искренне вырвавшихся из души моей, я помню это (29(2), 132-133)].13 Until recently this letter of Dostoevsky's was only available in a draft copy made by his wife, Anna Grigorievna, as reprinted in 1986 in Volume 29(2) of Dostoevsky's letters. But in 2020 the original autograph of the letter was discovered in the "Archives of the Office of the Successor Tsesarevich in the Ministry of the Imperial Court" and published in 2020 with some discrepancies between it and the earlier version made emphatic.14 One other letter from Dostoevsky to the Grand Duke Alexander Alexandrovich was also published for the first time in 1986 and dated 28 January 1872. It was a letter of thanks written after Dostoevsky's request for assistance in the earlier letter (now missing) delivered by Meshchersky (as discussed above) had been granted. Nothing was publically confirmed for over a hundred years following Dostoevsky's death about the assistance that the Grand Duke had accorded him, until this letter of thanks from Dostoevsky of 28 January 1872 expressing his gratitude was published for the first time in pss (29(1) 226). In his letter of thanks it is not entirely clear what "favour" precisely Dostoevsky is thanking the Grand Duke for. Perhaps this lack of clarity was deliberate on Dostoevsky's part because he was expecting this letter to be opened by the heir's secretary and did not wish to embarrass the Grand Duke for his magnanimity towards a former political convict? He did write however: "My feelings are vague: I am both ashamed of the boldness I exhibited, and at the same time I am filled now with delight at the priceless attention that Your Highness has paid to my request. It is dearer to me than anything else, dearer than the very help that You gave me and which saved me from a great calamity."15 [«Чувства мои смутны: мне и стыдно за бывшую смелость мою, и в то же время я исполнен теперь восхищения от драгоценного внимания Вашего высочества, оказанного просьбе моей. Оно дороже мне всего, дороже самой помощи, мне оказанной Вами и спасшей меня от большого бедствия (29(1), 226)»]. There were indirect rumours at the time that Dostoevsky may have received some financial assistance from the highest authorities, including hints contained in his own correspondence, though these were not confirmed and Anna Grigorievna never mentioned this in her own memoirs, although her descriptions of the financial hardships experienced by them virtually dominate her reminiscences. She writes of their debts after their return to Russia (i.e. 1871) and that: "with accumulated interest, they totaled twenty-five thousand by the time we returned from abroad. We had to pay them back for 13 years. Not until the year before my husband died did we settle them at last."16 The impression is created that the debts were paid off solely due to her astute handling of the creditors and the enterprises that she initiated to publish his works independently and supply them to booksellers and to subscribers directly. Finally this culminated in the opening of their new business at the start of 1880: "f.m. dostoevsky, bookseller to the provinces only".17 Yet Dostoevsky's own correspondence provides some evidence of the financial assistance that he received. For instance, he was also able to pay off a long-standing debt to his faithful friend from his younger days, doctor S.D.Yanovsky, who had cared for him whenever he had been unwell (29(1), 228-230) (although Dostoevsky ultimately politely rejected him). Dostoevsky also wrote to his favourite niece, Sonia Ivanova, with whom he had maintained a fairly intimate correspondence saying that he had received "a considerable amount of money" and was able to satisfy some of his creditors (29(1), . Sonia Ivanova's letters to Dostoevsky have never been found and appear to have been lost. In an article by Vera Torzhkova, who represents herself as a distant descendent of the Ivanovs, it is alleged that: "Correspondence with Feodor Mikhailovich ended shortly after Sophia's marriage. Dostoevsky's letters to Sophia were burned by the descendants of the Ivanovs as discrediting (!?) their family". [«Переписка с Фёдором Михайловичем оборвалась вскоре после замужества Софьи. Письма Достоевского к Софье потомки Ивановых сожгли, как порочащие (!?) их род»].18 Rumours had also been circulating in the late nineteenth century that Meshchersky had been receiving subsidies for his newspaper The Citizen from the Imperial budget due to his long-standing relationship with the Alexander Alexandrovich, who had been crowned by then as Alexander iii, after his father's assassination in 1881. But that was after Dostoevsky's demise. His widow was at pains to point out that Dostoevsky's involvement with The Citizen i.e. Meshchersky had turned many people against him. Dostoevsky was always very sensitive about any suggestion that he might have received some special favours from 'the powers that be' and the implication that this could have affected the content and thrust of his writings. In the third issue of The Citizen, 15 January 1873, in his column "Writer's Diary" following the assumption of his editorship one of the first things he made sure of was to dispel any such suggestions. He wrote in the chapter 'Something personal' [Нечто личное] (in connection with another topic): "But can anyone ever say of me that I sought or gained anything in that sense in any lieu whatsoever i.e., that I sold my pen?" [«Но когда и кто может сказать про меня, что я заигрывал или выигрывал в этом смысле в каком-нибудь lieu, то есть продавал свое перо» (21, 30) .] Yet could Dostoevsky's appeal to the heir for financial assistance not be interpreted other than in terms of Dostoevsky having "sold his pen"? Alexander Alexandrovich received copies of The Citizen and he would have had access to Dostoevsky's columns in 1873-74, including his "Writer's Diary". He may have seen another authorial commentary on Demons in the 50th issue The Citizen of December 10, 1873, 'One of Today's Falsehoods' , the last issue for that column, and Dostoevsky's last chance of writing a personal vindication of himself, and of his depiction of Russian society in Demons (21, (125) (126) (127) (128) (129) (130) (131) (132) (133) (134) (135) (136) . It encapsulates in a defiantly virtuoso performance of paradoxical and biodirectional argumentation the similarities in the political aspirations of young people to become Petrashevtsy, which he had shared, and which he now identified with the aspirations of young people to become Nechaevtsy, which he had exposed and vilified, all the while as he defends and attacks both trends and aspirations. For his part, Alexander Alexandrovich "attached great importance to Dostoevsky", and summed up Maria's legacy noting that it was from his mother that Dostoevsky inherited "an extraordinary emotional talent", one that might be described as a great matureness or fullness of the heart: ["именно от матери передалась ее сыну необыкновенная эмоциональная талантливость, -то, что хочется назвать развитостью сердца ... "]; Maria Feodorovna gave birth, raised and educated her children, was the "soul of home readings", "she was very gentle and tender", her portrait at the exhibition displayed her countenance "marvelous in its expressiveness and poetry" ["в экспозиции имеется ее портретизумительное по своей выразительности и поэтичности лицо".]24 The neglected piece of information that we would like to consider here are Andrei's impressions about his mother and her best friend Ekaterina. Yet these impressions are not confined solely to the cult of Maria's 'motherhood' centering on her children, that has been constructed in Russia in 'iconic' terms and promulgated as self-sacrificial, but in the context of her independent friendship with Ekaterina, who according to Andrei's belief was born into the British family of "Gardner": I will also mention Arkady Alekseevich Alfonsky and his wife Ekaterina Alekseevna, born Gardner Ekaterina Alfonskaya was a real friend of my mother's, and according to the stories of the latter, they saw each other almost daily. Andrei Dostoevsky is generally regarded as a dependable and trustworthy witness. But even if some details remembered by him are not entirely reliable, they always contain an element of truth. In this case he appears to have been convinced of the significance of his mother's friendship with Ekaterina Alfonsky and the English connection associated with her, and compelled to record it in his memoirs. However, he was wrong in his belief that she was born a "Gardner", for it was her sister, who was a Gardner, and her sister's family was known to the Dostoevsky family (which shall be discussed below). Ekaterina was at the time the wife of Arkady Alfonsky (1796-1869), who served as a physician from 1817 onwards at the Moscow Hospital for the Poor, renamed the Mariinsky Hospital, becoming from 1830 a consultant. At the same hospital from January 1821 onwards Dostoevsky's father Mikhail also served as a doctor. Both doctors' families resided for a period of time on the territory of the hospital (the Dostoevsky family resided in the left wing of the hospital from 1821 until just after the death of Maria in 1837), their children were of similar age and would have shared many experiences: "The five children of the Alfonskys' () together with the children of the Dostoevskys' wandered around in the large garden of the Mariinsky Hospital".26 After Ekaterina's death in September 1829, Alfonsky remarried. From 1819 onwards he also taught surgery at Moscow University, later was appointed Dean, then Pro-rector and from 1842 as Rector, a position he retained on and off for almost 20 years.27 The names of Alfonsky and members of his family are woven into the preparatory notes to Dostoevsky's intended major work Life of a Great Sinner [Zhitie velikogo greshnika]. Allusions to this family, tied up with incidents from Dostoevsky's own early life, reappear in the Notes to Demons, and are echoed in the text of The Adolescent and Brothers Karamazov. The most recently published edition of the preparatory notebooks (zapisnye tetradi) to Demons, in a 'diplomatic transcript' (diplomaticheskaia transkriptsiia) reproduces Dostoevsky's hand-written verbal version of the manuscript combined with the graphical in its exact arrangement on each page enabling one to see allusions to the Alfonsky family in the context of Dostoevsky's process of literary creation. It makes it easier to trace the genesis of the author's ideas over a period of time co-existing with other ideas bursting on to the space of the page creating meaning that is inseparable from all its adjacent verbal-graphical and calligraphic levels. New insights are gained into the allusions to the 'Alfonsky' name that becomes an important recurrent motif in the emerging narrative about the hero's teenage years in the context of Dostoevsky's own family superimposed onto to the Alfonsky family and intermingling with it. Sometimes the emblematic sign of the shortened form of "Alf-y" or "A-y" is used to cover up what seem like direct references to the life of Dostoevsky's own father Fedor and Dostoevsky's own boyhood, suggesting that for some reason he was reluctant to name his father openly even in his working notes. On the other hand, it also suggests that the two families' relationships and fates were closely intertwined, making Andrei's reminiscences all the more significant. The notes also contained the motif of friendship between two women, which was not included into the final text.28 In spite of some of Andrei Dostoevsky's claims about Ekaterina being incorrect in their detail, his memoirs, in the light of Ekaterina being Dostoevsky's mother's best friend (in today's idiom her 'Bestie'), as well as having an English connection via the well-known name of 'Gardner' , and the importance he attaches to the fact of the two friends being buried next to each other, need investigating further. By associating Ekaterina with the well-known 'Gardner' family, Andrei was referring to the descendants of the founder of the Gardner dynasty, Francis Iakovlevich (Jacob) Gardner (1714?-1796), who had arrived in Russia in 1746, eventually settled in Moscow and finally in early 1766 received permission from the College of Manufacturers to formalize the establishment of his porcelain factory.29 Not a great deal is known about "the Romantic Englishman" Francis Gardner, who apparently was a Scotsman and traded as a wood merchant in Nemetskaia street (now Baumanskaia street), also opened a banking business and ran a sugar refinery.30 He traveled a great deal around the Russian Empire, including Siberia, Ukraine and the White Sea looking for raw materials, such as kaolinite to manufacture porcelain and a suitable location for his enterprise. Eventually, in order to fulfill the Imperial government's requirements for the purchase of land (tied up with the possession of serfs), he purchased a site from Prince Nikolai Urusov in the Dmitrovskii district of the Moscow Oblast' , called Verbolovo. He remained a British subject never assuming Russian citizenship. In the early standard text of 1799 by William Tooke31 barely two pages are devoted to the Gardner porcelain business and he is mistakenly referred to as "Henry", though a longer account is available in a more recent study of Anthony Cross of 1997 , where all the available information was summed up, including that from Russian sources, which were also drawn on in the earlier publication by M.C. Ross.32 Cross points out that some confusion has arisen because "there was in St Petersburg at the same period another Francis Gardner of the English Club fame, whose father was also a James (d.1756), a treasurer of the British Factory since the early 1730s and owner of the rope-yard, who, to complicate matters even further, also had a brother named Francis (d.1755)".33 In recent years Gardner and his porcelain business have been attracting increasing interest on the web, though some of the details that have become available seem contradictory and questionable.34 It is evident that Gardner was not just a run-of-the-mill entrepreneur seeking his fortune in the Russian Empire, but may have had artistic pretensions being knowledgeable about European pottery and ceramics, not just British, but also that produced by the Sèvres factory in France (founded at Vincennes), as well as the Meissen factory in Germany near Dresden (famous for its Chinese vases), and the Du Paquier factory in Vienna, Austria. The success of these firms led to the establishment of other porcelain factories in Europe that propelled Gardner into porcelain production in Russia. Gardner made an unassailable name for himself when he presented a Gardner dinner set to Catherine II, and was then commissioned by her to fill an order for three more dinner sets for the Imperial Court between 1778-1785 comprising altogether four dinner services for celebrations to honour Knights of the Imperial Orders of St George, Andreevsky, Alexander and Vladimir that were held at the Winter Palace and other chambers on the feast days of the patron saints (ordenskie servizy).35 From what can be gauged from various geneological sites and encylopaedias, the Gardner porcelain factory was owned by several generations of Gardners from its foundation until 1892, when it was sold to the Old Believer merchant M.S. Kuznetsov.36 As for Ekaterina Alfonskaya, described by Andrei as a "Gardner" (who is featured with her husband Arkady and their family in Dostoevsky's Life of a Great Sinner), she is known to have had six children: Arkady (1819-1890), Ekaterina (Katia) (1821-?), Anna (1822-1888), Aleksei (1824-1872), Sof'ia (1827-?) and Liubov ' (1828-1901) .37 As noted above, she was not born a Gardner, as Andrei maintains, but her maiden name was Andreevskaya, and her patronymic was not Ekaterina Alekseevna, but Ekaterina Kirillovna. (Her father Kirill Samoilovich Andreevsky was a physician and professor of medicine at Moscow University, achieving the 7th rank in the service as 'nadvornyi sovetnik' . He had studied at the Kyiv military hospital and is listed in the available records as having been part of the Pereyaslav Collegium.)38 However, Ekaterina had a sister called Anna Gardner, who was married to a descendant of the Gardner family, Peter Frantsevich Gardner (b. 1791 os). In all of the references to do with Gardner porcelain Manufactory he is referred to as the son of founder Francis senior, (although Francis had an elder son, who died young at around the age of twenty, whose name was also Francis i.e. junior),39 and the factory had eventually passed on to him (Peter Frantsevich) and his brother. Anna Gardner and her husband Peter Frantsevich (also referred to as Garner) had a large family, who were first cousins to the Alfonsky children, and some of these Gardner sons were known to the Dostoevskys, including the older brother Mikhail, since they attended some of the same educational institutions. F.M. Dostoevsky definitely knew some of the Gardner youths, since in an early letter to his father, Mikhail, written on July 3, 1837 at St Peterburg when Dostoevsky (and his brother) was attending the preparatory boarding school of Koronad F. Kostomarov and preparing for their entry exams to the Main Engineering Academy, he mentions having seen 'Garner' (Гарнер) and another school mate, who had also attended the preparatory school, but had left earlier, and were already attending the Engineering Academy: "Last week we saw our former class mate Garner and Vessel. They visited Koronad Fillipovich (Kostomarov) to say goodbye, since they are leaving for a summer camp at Peterhof. Today is the birthday of K.F." (28(1), 36).40 Dostoevsky would have only mentioned these names if he had known that his father approved of these young men, since in his letters Dostoevsky tried not to upset or provoke his father in any way, though this was nevertheless more-often-than-not the case. Dostoevsky was accepted into the Engineering Academy in late 1837 and began studies in January 1838. When one checks the records of pupils at the Engineering Academy in the publication Historical Outline of the Development of the Main Engineering School 1819-1869 (1869)41 one discovers amongst the lists in the 'Attachment' (Prilozhenie) that there was a student by the name of 'Gardner' for the period when Dostoevsky was studying there: an "Alexander Gardner" was a pupil in the Lower Officer's Class of Warrant Officers or Ensigns (Iz nizhnego ofitserskogo klassa: Praporshchikami), which in the tsarist army was the most junior officer rank in 1841.42 (See attached illustration of the page for that year.) On August 20, 1841, Dostoevsky had sworn his 'Oath of Allegiance to Service' with 20 other warrant officers, who would continue being his classmates for another two years, who are described in detail in a recently published collection of available archival sources.43 Dostoevsky's teachers and class-mates are also discussed for the years 1837-1839 (as they had been in an closest friends at the Engineering Academy.45 Regarding this friendship, Joseph Frank in his biography of Dostoevsky goes so far as to suggest that "an element of latent sexuality may have been amongst its components", citing in his defence Dostoevsky's own letters.46 Another well-known name mentioned in Maksimovsky on the same page as that of Gardner (see illustration) is Fedor Radetsky (1820-1890), who graduated in 1840 and became an adjutant-general and a 'hero' of the Russo-Turkish war. Dostoevsky attended a celebratory dinner in his honour in 1878 in St Petersburg, and gave a short speech. 47 (31(1), 22) . Over 150 former students of the Engineering Academy were present at that dinner, according to newspaper reports, and had Alexander Gardner been in St Petersburg, he is likely to have attended that dinner too (31(1), 80). Information about the Gardners has been difficult to find and to verify in New Zealand, away from the archives in the Russian Federation. The available records on websites such as geni, Rodovod, Ancestry etc,48 as well as specialist sites like Russian Military Engineers or the Ryazan' District,49 or documents about Noble Families of the various regions in the Russian Empire (including ennobled families) of the Moscow region and Ryazan' etc show many discrepancies regarding the years of birth, death, family relationships and ownership of the Gardner Manufactory etc, which is also reflected in the available articles. But it has been possible to determine that the sister of Ekaterina, Anna Kirillovna Gardner (Andreevskaya), the wife of Peter Frantsevich Gardner, gave birth to ten boys, of whom several died in infancy, but about six survived into adulthood. Of these three attended the Main Military Engineering Academy graduating from the Conductor's classes in 1836, 1838 and 1840 respectively, which Dostoevsky also completed in 1841. But he continued with his studies graduating from the officer classes of the Main Institute as a lieutenant in August 1843 and was released as a field engineer. The eldest of the Gardner boys and the first one to attend the Engineering Academy (before Dostoevsky had enrolled) was Petr Petrovich Gardner (1816 N.S.-1881).50 He is described as a military engineer who had been decorated by the time he retired with a St George Cavalier award, achieving the rank of lieutenant general. Earlier, as part of the troops of the Peterhof detachment (since 3.1854) he participated in the protection of the coast of the St Petersburg province and earned the Tsar's "Highest Favour" for his work on the "installation of batteries at the mouth of the Neva". (Would these have been coastal fortifications, or could these have had something to do possibly with the installation of telegraph transmission taking place at the time, which required porcelain insulators?) He took part in the defence of Sevastopol. He is buried at the prestigious cemetery of the Novodevichy Monastery in St Petersburg, founded in 1849, where noble families, prominent statesmen and notable figures were interred.51 Perhaps the best known of the Gardners to have attended the Military Academy was Vladimir Petrovich Gardner (1819-1886), who graduated in 1838 from the Conductor's classes of the Main Engineering Academy. He was released as a Junker.52 In 1846 he was seconded to the Moscow Gendarme Division and was staff captain. In the 1850s, together with his brother Alexander, he became the owner of the Gardner Manufactory in Verbilki. He died and was buried in Moscow. What is surprising is that he is buried at Lazarevo cemetery in the same plot as Ekaterina Alfonsky, Maria Fedorovna's friend. (This will be discussed in greater detail below.) The youngest Gardner brother to attend the Engineering Academy was Alexander Petrovich Gardner (4.11.1820 -?), whom Dostoevsky definitely knew. He enrolled in the Conductor classes and graduated in 1840, but remained at the Academy to continue his education. In 1841 he graduated from the officer class and was released as an ensign to field engineers. He served in the Life Guards Sapper Battalion, a separate part of the Russian Imperial Guard, stationed in St. Petersburg. He retired in 1847. In the 1850s together with his brother Vladimir, he became the owner of the Gardner Manufactory in Verbilki.53 The other Gardner boys' names were Pavel (b. 1826 -1869), Sergei (b.? -1874), Konstantin (1834 -1898) (as well as four others: Aleksey, Mikhail, Viktor and Nikolai, who did not survive past infancy). The older Gardner lads would have been likely to visit their aunt, Ekaterina at the Mariinsky Hospital, as may have another descendant of Francis Gardner's (described by commentators as his son, Nikolai (born 1798?-died 1846), who had six children, second cousins of the Alfonskys.54 Nikolai retired from military service with the rank of lieutenant in 1826, and was the first member of the Gardner family to be ennobled. He lived at Verbilki, where he proceeded to learn four foreign languages and acquired a 'rich' (bogataia) library.55 One wonders whether he lent his books to other members of the family? This also begs the question as to whether members of the Gardner family may have suggested various educational establishments to Dostoevsky's father, when the latter was deciding on where to send his sons to further their education, including the various preparatory and boarding schools (Pansiony) that the Dostoevsky brothers attended, at some of which the older Gardner brothers appear to have studied earlier. These include the N.I. Drashusov (Souchard) Day school and the Leonty (Leopold) Chermak Boarding School in Moscow, and in St Petersburg the Konorad Kostomarov Preparatory Boarding School, where Dostoevsky saw Alexander Gardner (28(1), 36). The Gardners seem to have attended the Chermak Boarding school, (as had Arkady Alfonsky apparently), although not at the time when Dostoevsky and his older brother Mikhail studied there. English was taught at that boarding school and the English tutor's name was "Asppel",56 (which is reminiscent of the name of the English character in Dostoevsky's novel The Gambler (Igrok), whose surname is Astley). One of their classmates at Chermak's was an English student George Harvey,57 whose father, Edward Harvey (1798 Harvey ( -1874) ) In the early 1830s amongst students attending his classes were M. Lermontov and V. G. Belinsky.59 How would Dostoevsky's father have made the decision to send his sons to the Saint Petersburg Engineering Academy, known at the time as the Nikolaevsky Engineering Academy that had only been established in 1810 under Alexander I, unless he had consulted someone informed about the education available at this new institution? Hence the role of the Gardners cannot be entirely discounted from Dostoevsky's life, nor can the role of the latter's cousins, the Alfonsky children. In addition there is the confusion between the Alfonskys and the Gardners reflected by Andrei in his memoirs, who must have had some reason for believing that the Alfonsky children were descended from the Gardners on the mother's side. It has not been possible to find any reference to the 'Gardner' connection in any works about Dostoevsky published in English, and only two references in Russian sources (as far as it has been possible to ascertain), both sourced from Andrei's memoir. In Joseph Frank's five-volume biography of Dostoevsky there is no mention of the names of Alfonsky or Gardner,60 neither are these names mentioned in the first volumes of the recent "exhaustive" biography by Thomas Gaiton Marullo.61 The two Russian references include the biography of the early years of Dostoevsky by Nina S. Nechaeva, the archivist and researcher of Dostoevsky's manuscripts and of his surroundings, and related (like Maria Fedorovna by birth) to the Nechaev branch of the family. N.S. Nechaeva directly cites Andrei Dostoevsky's reminiscences in her work The Early Dostoevsky 1821 -1849 (Rannii Dostoevskii. 1821 -1849) , which is later repeated in the first volume of the Chronicle of Dostoevsky's Life (Letopis' zhizni i tvorchestva F.M. Dostoevskogo) compiled by Irina Yakubovich.62 N. S. Nechaeva mentions in a footnote in her book that she visited the Lazarevo cemetery before that cemetery was destroyed and the graves were liquidated and disinterred: "Before the liquidation of the Lazarevo cemetery at the end of the 1920-s when I tried to arrange to have the grave of Maria Fedorovna Dostoevsky moved into the Museum of Dostoevsky, I wrote down the inscriptions on the In the illustrations that accompany the recent article about the restoration of Maria Fedorovna's tombstone, Pavel E. Fokin, Director of the Russian Literary Museum in Moscow includes a copy of a drawing from the Museum's archives of a rough plan of the original location of the Maria's grave at the Lazarevo cemetery that must have been done just before the graves were exhumed in the 1930s. In the drawing her grave has "M.F.D." written over it, while an area right next to it is a family plot for the Gardners, at least four times the size of the plot for Maria Fedorovna, which is identified with the names of "Alfonskaya and Gardner" written over it.65 This illustration is being reproduced here with acknowledgement (Illustration). According to the three-volume Moscow Nekropolis (1907) several members of the Moscow Gardner family were buried at Lazarevo cemetery in the vicinity of Maria Fedorovna's grave. These are identified in the Nekropolis as being in addition to Anna Gardner neé Andreevskaya, listed independently with no dates, also "Gardner, Vladimir Petrovich b. exclusive cemetery of the Alekseevsky convent built in the name of 'Alexis, the man of God' . The Alekseevsky convent was the oldest in Moscow dating back to the 14th century. (It was transferred in 1837 from its original site and rebuilt in stone in Krasnoe Selo, with its church consecrated in 1853, and the Church of All Saints built between 1887 and 1891.)69 What is surprising is that there is no mention of the British name of Gardner in the account of Dostoevsky's Moscow years by G.A. Fedorov, (who was the first to propose that Dostoevsky's father was not killed by his serfs, but died a natural death). Fedorov does cite Andrei's memoirs and expands on them, showing that Dostoevsky's mother's friend Ekaterina was apparently in an emotionally abusive relationship perpetrated by her husband, Arkady A. Alfonsky: Maria Fedorovna, the mother of Dostoevsky had some beloved graves at the Lazarevo cemetery that were of her mother and father, but as she was dying she 'ordered' (povelela) that she be buried next to the grave of Ekaterina Kirillovna Alfonskaya. Dostoevsky's mother was friends with the first wife of Alfonsky, as Andrei Dostoevsky recalls, they met almost daily. And Fedor as a boy knew well the sons of Alfonsky from his first marriage, Arkady and Aleksei, and the daughter Katya. Alexei's fate will pass before Dostoevsky's eyes in the 1820s and the 1830s, and then later too after his return from Siberia, when he would live in Moscow in 1864 almost for the whole year./ And later too he used to visit Moscow and while visiting his mother's grave he would walk past the graveyard monument with the inscription 'E.K. Alfonskaya, born Andreevskaya' ./ We do not know how the marital relationship between E.K. and A.A. Alfonsky developed. We only know of the evidence supplied by the chief physician Kh.I. Oppel about the "cruel deeds" (o zhestokikh postupkakh) perpetrated by Arkady Alekseevich towards his "first wife".70 Andrei Dostoevsky would have passed those graves of the Gardners with that of Ekaterina Alfonsky at Lazarevo cemetry when he visited his mother's grave whenever he was in Moscow, since he spent virtually his entire working life as architect (from 1849 to 1890) serving in Ukraine, which was also known in the Russian Empire as Little Russia (Malorossiia). He designed some important buildings in Elizavetgrad, Yekaterinoslav, Simferopol, and also the Russian town of Yaroslav. Today the first two towns are known as Kropyvnytskyi in the Kirovohrad Oblast' , while Ekaterinoslav is now known as the city of Dnipro, that was formerly Dniepropetrovsk. Simferofol has retained its name as the second largest city in Crimea. Andrei moved back to Russia shortly after his retirement in 1890 and completed his memoirs around 1894, two years before his death. He may have shown them to Dostoevsky's widow, Anna Grigorievna. But the memoirs were first published in 1930 by his son, by which time Anna had been dead for some years (d. 1918) . Andrei had a fairly distant relationship with Fedor Dostoevsky and appears to have almost hero-worshipped him. Dostoevsky's other siblings were more critical of their famous brother, despite the accepted narrative that Fedor Dostoevsky's older brother Mikhail (1820-1864) had an ideal brotherly relationship with Fedor. Yet Andrei's reminiscences remain unsurpassed in the context of the boys' childhood and adolescent years. What were the reasons for Andrei Dostoevsky pointing out his mother's close relationship with Ekaterina Alfonsky and associating the latter with an English connection identified with 'Gardner'? There may be more to this Gardner linkage than we know of today. It might also suggest that the interaction between the two friends might have been due to them having interests in common. Maria Dostoevskaya loved poetry, literature and music. She is reputed to have been a voracious reader of novels and very musical, able to play the guitar. She is credited with having inspired in her sons a love of poetry and instilled in them an interest in literature and music. Her remaining fairly heartwrenching nine letters to her husband are written in beautiful handwriting in the 'sentimental' mode prevalent at the time, and reveal a poetic and emotionally responsive young woman.72 All the Dostoevsky boys apparently adored their mother and the two eldest composed the poetic epitaph for her tombstone.73 Her undeniable influence on them and her own personal friendships merit attention. One wonders at the extent of her guidance on the selection of books that were available in the Dostoevsky household, since her grand-daughter Liubov' (Dostoevsky's daughter) specifically mentions Maria's involvement in her son's reading, information that could only have come to Luibov's directly from her father. One cannot help wondering why Dostoevsky in his childhood seems to have had so much access to literature translated from the English and written from a Protestant perspective, as demonstrated repeatedly in Andrei's memoirs and in the recollections of Dostoevsky himself, some of which we are all familiar with.74 It's usually explained solely in the context of it being literature that was available and popular at the time, such as Gothic fiction, epistolary and sentimental novels exemplified by Richardson's Pamela; or Virtue Rewarded, The Vicar of Wakefield by Oliver Goldsmith, historical adventure stories, such as the numerous Waverley novels of Sir Walter Scott, the novels of Dickens, especially those featuring children and adolescents, or those expressive of 'humorous idealism' , Sterne's Sentimental Journey, Defoe's Robinson Crusoe, also Fenimore Cooper's stories set amongst the native or indigenous populations of North America, and later also Byron and Shakespeare, Maturin, de Quincey, Thackeray, Brontë, etc. Such literature does not sit well with the known stern temperament of Dostoevsky's father. Even the first stories from the Bible that Dostoevsky was introduced to were translations from the Protestant version of the Bible as narrated by the German writer Johann Huebner (1668-1731).75 Could there have been a connection between the literature favoured by his mother and available to the Dostoevsky household at Mariinskii Hospital, literature that was perhaps also favoured by her closest friend Ekaterina? Could Dostoevsky's family's readings in the evenings before bedtime, at a time when he could not yet read himself and would remember for the rest of his life have had any input from the mutual literary and artistic interests of the two women friends? It seems that Dostoevsky's introduction to and love for much British literature was formed during childhood and indirect evidence suggests that his mother's friendship with Ekaterina is likely to have facilitated Dostoevsky's access to such literature. We all remember how in Winter Notes on Summer Impressions Dostoevsky recalls that "even as a small child, when still unable to read, I listened agape, enthralled and terror-struck in turn, to my parents' bed-time reading of Mrs Radcliffe's novels, which put me into a fever and kept me awake at night" (V, 46).76 His other dominant memory is of the novels of Sir Walter Scott, corroborated by Andrei, who recalls in his Memoirs that he saw in Dostoevsky's hands most often the novels of Sir Walter Scott, especially Quentin Durward and Waverley. The older brothers had their own copies and reread them several times, despite the ponderous and dated translation.77 Dostoevskii recalled many years later on 18 August, 1880 while responding to a query about suitable reading for children that would inspire their imagination, that he himself, at the age of twelve, in the countryside during vacations, had read all of Walter Scott, and it had developed his own imagination and sensitivity: "Walter Scott has a lofty educational significance", and in this context Dostoevsky also recommended all of Dickens "without exception" (30(1) 212). An example of their mother's indulgence towards her children is reflected in Andrei's recollections of the games the boys used to play during the summers they spent at Darovoe from 1832 onwards, their newly purchased estate in the country side in the Kashirsky district of the Ryazan province. Here they were under the sole care of their mother, while their father remained working in Moscow. Fedor invented the game of Robinson, with him playing Robinson, while Andrei had to impersonate Friday.The other games they played were derived from adventure stories about the "tribes" of indigenous populations of North America that seemed to have been based on the stories of Fenimore Cooper that were translated into Russian from the late 1820s onwards.78 As Pushkin had noted: "Chateaubriand and Cooper, both introduced us to the Indians from their poetic side and painted over the truth with the colors of their imagination" [« Шатобриан и Купер, оба представили нам индийцев с их поэтической стороны и закрасили истину красками своего воображения»]79 Andrei recalls some of these games: Brother Fedya, who at that time already read a lot, probably got acquainted with the description of the life of savages. "Playing being wild" was our favorite game. It consisted in the fact that, having chosen a denser place in a linden grove, we built a hut there, covered it with brushwood and leaves and made the entrance to it imperceptible. This hut became the main residence of wild tribes; (we) stripped naked and painted our bodies with paints in the manner of a tattoo, made waist and head ornaments from leaves and dyed goose feathers and, armed with homemade bows and arrows, made imaginary raids on Brykovo, where, of course, peasant boys and girls were found deliberately placed there. They were taken prisoner and kept, until a decent ransom, in the hut. Of course, brother Fedor, as the one who invented this game, was always the main leader of the tribes Of particular interest in this game was that we, the "wild ones", were not looked after by the elders and, in this way, completely secluded from everything ordinary -that was not wild. [Брат Федя, тогда уже много читавший, вероятно, ознакомился с описанием жизни дикарей. "Игра в диких" и была любимою нашею игрою. Она состояла в том, что, выбравши в липовой роще место более густое, мы строили там шалаш, укрывали хворостом и листьями и делали ход в него незаметным. Шалаш этот делался главным местопребыванием диких племен; раздевались донага и расписывали себе тело красками на манер татуировки, делали себе поясные и головные украшения из листьев и выкрашенных гусиных перьев и, вооружившись самодельными луками и стрелами, производили воображаемые набеги на Брыково, где, конечно, были находимы нарочно помещенные там крестьянские мальчики и девочки. Их забирали в плен и держали, до приличного выкупа, в шалаше. Конечно, брат Федор, как выдумавший эту игру, был всегда главным предводителем племен Особый интерес в этой игре был тот, чтобы за нами, "дикими", не было присмотра старших и чтобы, таким образом, совершенно уединиться от всего обычного -не дикого.]80 Their mother's indulgence and encouragement of games is further illustrated by her consent to let the children prolong their games by deciding not to call them in to dinner, as she "ordered the savages to take their dinner in the air in a special dish and put it somewhere under the bushes. This gave us great pleasure, and we ate dinner without the help of forks and knives, but simply with our hands, as befitted the wild ones." [«маменька, желая продлить нашу игру и наше удовольствие, решилась не звать нас к обеду и велела отнести дикарям обед на воздух в особой посуде и поставить его где-нибудь под кустами. Это доставило нам большое удовольствие, и мы съели обед без помощи вилок и ножей, а просто руками, как приличествовало диким"].81 Dostoevsky's love for adventure stories set amongst indigenous tribes of America continued, as Dmitry Grigorovich, who attended the Engineering Academy with him and later also shared lodgings recalled in his memoirs that Dostoevsky advised him to read various translations, including Cooper's Pathfinder or The Inland Sea (as well as Scott's Astrologer, de Quincy's
Confessions of an English Opium Eater etc).82 To get back to the crucial point of our discussion, namely the connection with the Gardners' clan in Moscow that arose due to the founder's establishment of the porcelain factory in Verbilki, it might be of interest to mention that Dostoevsky appears to have had a genuine interest in pottery, a love of beautiful ornaments and a special sensitivity towards it reflected both in his life and his works. One need only recall the observations in his widow's memoirs that when she first arrived at Dostoevsky's apartment to work as a stenographer she noticed two large Chinese porcelain vases decorating the window sill of his lodgings that he had been given by his friends in Siberia and had brought back all the way to European Russia: "The window sills were adorned by two large beautifully shaped Chinese vases."83 While living in Dresden she recalls Dostoevsky spending his hard-acquired cash at an auction because he could not resist buying some Florentine bowls: "Wouldn't it be nice to buy these lovely bowls? What about it, Anechka, shall we buy them?"84 After their return to Russia, pride of place in their modest apartment was a Meissen porcelain lamp that Dostoevsky had brought back from Dresden, plus a matching ashtray and "other trifles' , as described in the reminiscences of Anna's school friend, Maria Nikolaevna Stoyunina (1846 Stoyunina ( -1940)) .85 We all remember the symbolic significance of the Chinese vases in The Idiot, and the numerous motifs to do with porcelain and pottery in the novel (8, 189; 409; 433) . Or Dostoevsky employing the visual image of a paradoxical Golden Age in his Writer's Diary through the medium of decorated porcelain cups. But what I found to be of special interest was quite a sophisticated article on ceramics and porcelain in The Citizen, during his editorship at a time when he was in sole charge over the summer months and entirely responsible for commissioning and selecting articles for publication.86 It is an unsigned article written by someone quite knowledgeable about pottery and the plastic arts. It appears to be a translation from the German of a commentary on the displays at the World Exhibition in Vienna, the Austro-Hungarian capital being held at that time. It would have been translated into Russian by the critic N.N. Strakhov working for Dostoevsky at the time.87 All, or the majority of these reviews of the Exhibition in Vienna are likely to have been written by Max Schlesinger (1822-1881), and published in newspapers abroad that Dostoevsky had access to at his editorial office, since The Citizen's publisher subscribed to some foreign periodical press. Schlesinger used to contribute articles to the Augsburg Allgemeine Zeitung, the Kölnische Zeitung, the Brussels Indépendance Belge, and amongst others he was also the correspondent of the New Free Press in 1869-74, and for the Berlin National Newspaper. The article in The Citizen on exhibits at the 1873 Vienna World's Fair (Weltausstellung 1873 Wien) was placed at the beginning of the issue, following the column 'Moscow Notes' , reflecting its relative importance. It had been sub-edited and contained sub-headed sections on topics including the following: Italian marbles and majolica; The focus of the German exhibition; Berlin and Meissen porcelain; Something in general about pottery; Something about the discovery of porcelain soil in Saxony, France and other places; Majolica and faience.88 It provides a short history of the discovery of special clays, the fact that porcelain owed its developemnt in Germany and France to a series of chance-accidents: "Porcelain is now being made everywhere to greater or lesser perfection, as the exhibition shows. For kaolin is everywhere, and where special varieties of it are needed, they are delivered there without any difficulty. (So even today I heard from Mr. Siemens that kaolin for Russian factories is delivered from Cornwall)." [«В настоящее время фарфор делается повсюду в большем или меньшем совершенстве, как это показывает выставка. Ибо каолин находится везде, а где нужны особые его сорта, туда они доставляются перевозкою без всяких затруднений. (Так еще сегодня я слышалъ от г. Сименса, что каолин для русских фабрик доставляется из Корнуалля)".]89 The reason Siemens is quoted in the article in The Citizen regarding the availability of kaolin in Russia for the porcelain industry, is because the Siemens industrial manufacturing company had been contracted by the Russian Tsarist government already in 1853 to construct a telegraph network in the Empire, and the construction of telegraph wires used hundreds of thousands of porcelain insulators. Initially Siemens constructed a telegraph line from Warsaw to Russia's border with Prussia. After this a telegraph network was extended to cover the European part of the Empire, connecting St Petersburg, Moscow, Kyiv, Kovno (Kaunas), Revel (Tallinn), Helsingfors (Helsinki), while in the section of the Petersburg-Kronstadt line, the cable was laid along the bottom of the Baltic Sea. The owner-manager of the Gardner porcelain factory in Verbilki in June 1873, when the above article was published in The Citizen appears to have been the daughter-in-law of Anna Gardner (sister of Ekaterina Alfonsky, Dostoevsky's mother's closest woman friend). She was Elizaveta Nikolaevna neé Leman (1838 Leman ( -1913)) , the widow of Pavel Gardner (1826-1869), who was the last one to assume ownership of the factory after the retirement of the older Gardner brothers. She may have visited the Vienna World Fair and viewed the porcelain exhibits, and may have even contributed samples from the Gardner Verbilki factory to the Russian displays, although the majority of exhibits from the Russian Empire at the time consisted mainly of exhibits from the State's Imperial factories, rather than private enterprises. Elizaveta Gardner is buried in the cemetery of the Ascension Church in Novo-Nikolskoe near Verbilki, where some other members of the Gardner family are also buried.90 -hand (27, 384) . She had apparently indicated that the note was intended for an "editor of a newspaper", although she never specified which particular newspaper or editor. The existence of the biographical note was confirmed in the first biography of Dostoevsky compiled by O.F. Miller for the posthumous edition of his works in 1883, where he noted that a biographical note had been dictated by Dostoevsky "a few years prior to his death".92 Dostoevsky had been displeased with the biographical entry about him by V.R. Zotov that had appeared in the Russian Biographical Dictionary (Russkii biograficheskii slovar') in 187593 and expressed his displeasure in the January issue of Writer's Diary [Dnevnik pisatelia] for 1876.94 Soon after (1876-1877) he was asked to provide his biography by at least two other contemporary literary biographers: P.V. Bykov and P.N. Polevoi. He promised to do so, but apparently (Leningrad: Nauka, 1972 -1990 ), 22 (1981) , 37-38. Hereafter references to this edition, pss, will be given in brackets within the text, citing volume and page number. Quotations in English translation will not be provided with their originals in Russian for reasons of space. did not fulfil his promise. But then in 1877 an illustrated volume of 50 very short biographies of Russian writers was published: 'Silhouettes' of fifty Russian literati and their short biographies ("Siluety" piatidesiati russkikh literatorov i kratkikh ikh biografii). It represented Dostoevsky as a "writer-psychiatrist" (pisatel'-psikhiatr), whose novels were "wonderful in their psychological analysis of character". The entry contained some minor inaccuracies.95. Also in 1877 he was approached by the editor of the Illustrated Newspaper (Illiustrirovanniia gazeta) to provide his photograph and a list of his works for inclusion in a supplement-collection of Modern Russian Celebrities (Sovremennye russkie deiiateli), which appeared in December of that year.96 The publication of these biographies may have contributed to motivating Dostoevsky to dictate his own biographical note to his wife.97
C When republished in the 30-volume pss, it was accompanied by an explanation from the editors in which it was stated that the note had been composed probably at the request of some "French foreign correspondent". As they explained, the dictated version contained the word 'Revue' , considered to be a French word for a journal, though it wasn't noted that the equivalent English word 'Review' sounded, when dictated in Russian, quite similar to the French 'Revue':98 "However, soon, probably at the request of some French journalist, Dostoevsky dictated to his wife the published biography." [Однако вскоре, вероятно по просьбе какого-то французского журналиста, Достоевский и продиктовал жене публикуемую биографию.] (27, 385) There has never been any reference as to the identity either of the "editor of a newspaper", nor of the "foreign correspondent" for whom the note was said to have been intended in the above-mentioned earlier publications in Russia. It is our intention in the current study to speculate as to the identity of these possible recipients of Dostoevsky's version of his autobiography. Since the pool of possible recipients is a very wide one, we are proposing in this instance to leave 95 "Siluety" piatidesiati russkikh literatorov i kratkikh ikh biografii, (S.-Peterburg: Izd. A Chernokhvostovoi, 1877) 50. 96 Letopis ', vol. iii, 200, 204, 238 discussion of possible editors of Russian-language newspapers to colleagues in Dostoevky's home country, and to focus instead on possible editors of foreign language newspapers and foreign correspondents, both French and British. It may be worth noting that shortly before the time when Dostoevsky dictated his biography, he had been in correspondence with Ignaty Horn, the editor of the French-language St. Petersburg newspaper, affiliated to the Russian Foreign Office, Journal de 318, 334) . Horn had asked Dostoevsky for permission to publish his short-story Krotkaia (The Meek One) in French translation in his Journal, to which Dostoevsky agreed. In the previous year (1876) Horn had arranged some receptions for George Augustas Sala (1828 Sala ( -1895)) , (formerly contributor to Household Words. A Weekly Journal conducted by Charles Dickens), who was revisiting Russia as a foreign correspondent and looking for material on popular cultural life.99 Horn may have had an interest in a short biographical sketch of Dostoevsky either for his own use, or for dissemination to some foreign correspondent (like Sala). However, we are proposing a more likely hypothesis that the autobiographical note may have been drafted on the advice of Konstantin Pobedonostsev, senator and a member of the State Council, who knew some British and French foreign correspondents working in St Petersburg, and in addition corresponded with a number of these and others. This was the case especially in 1876 prior to the commencement of the Russo-Turkish war on 24 April 1877 and lasted until after the war ended on 3 March 1878. During that period, Pobedonostsev translated from English some publicist works on the Balkan crisis and reviewed a number of books and pamphlets by British authors he was in contact with. He and Dostoevsky used to meet fairly regularly throughout the period when the autobiographical note was meant to have been composed, ie a few years prior to Dostoevsky's death and before he had begun his intensive work on the composition of The Brothers Karamazov.100 99 Sala, George Augustus, The Life and Adventures of George Augustus Sala, (London, Paris, & Melbourne: Cassell and Co. Ltd, 1895), vol. ii, 317-318: "I was most cordially received by the Ambassador, who, among other favours, introduced me to a clever gentleman named Horn, who was the editor of the Journal de St. Pétersbourg, a daily paper shrewdly suspected to be the organ of the Russian Foreign Office, and published in the French language () Mr. Horn was kind enough to hold at his residence a couple of receptions at which I had the honour to meet, so I was given to understand, the flower of intellectual Russian society. One gathering was composed exclusively of university professors and medical men. In 1876 I had, apparently, hopelessly-but such turned out not to be the case-forgotten all the Russian which I had acquired in 1856; so the "medicos" and the professors all talked to me in French." 100 Grossman, 'Dostoevskii i pravitel'stvennye krugi 70-ykh godov' , Prilozheniia. I. Pis'ma K.P. Pobedonostseva, 124 -142. The British foreign correspondent for The Times of London from 1877 to 1878, Sir Donald Mackenzie Wallace (1841 Wallace ( -1919) ) may have been a possible intended recipient of Dostoevsky's autobiographical note. He was in St Petersburg throughout 1877 until he was dispatched to cover the Berlin Congress in June-July 1878. He had published a two-volume book entitled Russia in January 1877,101 which was translated almost immediately into French,102 but held up by censorship in Russia.103 In his book Russia Wallace writes: "In March 1870 I arrived for the first time in St Petersburg. My intention was to spend merely a few months in Russia, but I unexpectedly found so many interesting subjects of study that I remained for nearly six years -till December 1875 Since my return to England I have kept up a constant correspondence with numerous Russian friends, so that I have been able to follow closely what has taken place in the short interval".104 Wallace met many prominent Russians and consulted them about various topics that interested him. In the Preface to his book he thanks various personages he was indebted to. These include "the late Mr. N.A. Milutine, the late Mr. Samarin, Prince Tcherkassky, and Mr. Koshelef", as well as a number of others such as "Madame de Novikoff, née de Kireeff", who resided in London. There were many other Russians he was acquainted with, but does not mention directly in his book. According to Pobedonostsev's biographer Robert Byrnes, both Pobedonostsev and his friend Catherine Tyutchev had "spent long evenings with Wallace between 1875 and 1877, correcting what they considered his errors in understanding Russia, especially the character and role of the Church."105 Pobedonostsev approved more-or-less of Wallace's assessment of social and political life in Imperial Russia, as did other conservative-minded Slavophile orientated Russian patriots at the time, such as I.S. Aksakov (though his opinion may have changed after the Congress of Berlin). In his book Wallace refers to Ivan Aksakov in the following terms: "I have long known Mr. Aksakoff, and have never in any country met a more honest and truthful man".106 Dostoevsky and Wallace first met briefly at the house of Aksakov and his wife Anna (sister to Catherine Tyutchev) in Moscow between 1873-74. Aksakov recalls this meeting in passing in a footnote that he attaches to an article by Apollon Maikov in Aksakov's journal Rus' after Dostoevsky's death. He mentions that Wallace had been in Russia for about three years by then, which would date it around the end of 1873, as Wallace had arrived in Russia in 1870:107 Once, while passing through Moscow, Dostoevsky came to see us and talked with enthusiasm about the late Sovereign Nikolai Pavlovich (Nicholas I -I.Z.), about how the historical image of the monarch, who believed in his dignity and his right, is majestically drawn against the backdrop of the past, and how sympathetic this image is to him. During the conversation, the famous English traveller Wallace Mackenzie came in; had lived in Russia for three years, had learned Russian very well and was familiar with Russian literature. When he learned that Dostoevsky was in front of him, he became curious and eagerly began to listen to Fedor Mikhailovich's interrupted and then resumed speech about Nikolai Pavlovich. Dostoevsky went on talking, paying no attention to the Englishman, and soon afterwards left. "You say that this is Dostoevsky?" the Englishman asked us, -"Yes." -"The author of The House of the Dead?" -Yes, that's the one. Well, what then? -"But how can he praise the man who exiled him to hard labour?" -It is difficult for you foreigners to understand this, we answered, but for us it is quite clear, as it's a completely national trait Ed. [«Как-то раз, проезжая через Москву, Достоевский зашел к нам и с увлечением разговорился о покойном государе Николае Павловиче, о том, как на фоне прошлого величаво рисуется этот исторический образ монарха, верившего в свой сан и свое право, и как сочувствен ему этот образ. Во время разговора вошел известный английский путешественник Уоллес Мэкензи, проживший перед тем уже года три в России, отлично выучившийся по-русски и знакомый с русскою литературою. Когда он узнал, что перед ним Достоевский, он загорелся любопытством и с жадностию стал слушать прерванную было и снова возобновившуюся речь Федора Михайловича о Николае Павловиче. Достоевский продолжал говорить, не обращая внимания на англичанина и вскоре затем уехал -Вы говорите, что это Достоевский? -спросил нас англичанин. -Да. -Автор «Мертвого Дома»? -Именно он. -Не может быть. Ведь он был сослан на каторгу? -Был. Ну, что же? -Да как же он может хвалить человека, сославшего его на каторгу? -Вам, иностранцам, это трудно понять, отвечали мы, а нам это понятно, как черта вполне национальная Ред.»]108 It was typical of Dostoevsky to have praised Nicholas I to specifically Aksakov, a Slavophile, who became a leading Pan-Slavist, rather than to someone left-leaning, for Dostoevsky had the ability to sense what the person he was speaking to or writing for would appreciate hearing at the time, and could attune himself to it. Similarly, in his autobiographical note he is likely to have attuned himself to whoever was the intended recipient of the note -of course, these are generalisations and one should qualify them by pointing out that sometimes the complete opposite happened. Wallace obviously gained special understanding about Slavophilism from his acquaintance with Aksakov and other Slavophiles, which is reflected in his chapter "Moscow and the Slavophils" in the first edition of his book Russia, being possibly the first wide-ranging critical account of Slavophilism then available in English. In Russia Wallace is considered to have contributed to the Russophile trend that developed towards the end of the century in Britain leading to the English-Russian Convention of 1907. (However, we would not go as far as this, as his assessment of Russian life, no matter how faithfully and affectionately it reproduces the ideal aspirations of society and people, is always combined with a healthy dose of humour and scepticism, even a sense of the absurd). As an ideology, Slavophilism and Russian Orthodoxy (with all its unintended offshoots) became a fashionable object of inquiry amongst some elements of British society, a trend that Wallace doubtlessly did contribute to, and which resulted in it also gaining more significance at home in Russia. The version of Wallace's book that is mostly reprinted today is an abridged version that includes about two-thirds The possibility that the foreign correspondent for whom Dostoevsky's note was intended may have been French has also been investigated in the course of our present study. The two most prominent French foreign correspondents at the time, both contributing at various times to the Revue des Deux Mondes were Anatole Leroy-Beaulieu (1842 -1912) and Alfred Rambaud (1842 Rambaud ( -1905)) . Pobedonostsev was acquainted with Leroy-Beaulieu, who referred to him as "The Russian Torquemada".111 Leroy-Beaulieu acknowledges in a Preface dated April 1881 to the first edition of his book L'Empire des tsars et les Russes that it was written over a period of eight years: "The work, of which I am offering the first volume to the public, is the fruit of ten years of work and four visits to Russia, from 1872 to last summer."112 From 1873 onwards Leroy-Beaulieu published articles on the Russian Empire in the Revue des Deux Mondes that were later included into the three volumes of his book. Alfred Rambaud in his book The History of Russia from its Origins until 1877113 actually mentions Dostoevsky's name (Dostoiévski) amongst a list of writers publishing "novels of manner".114 Rambaud also lists the name of Meshchersky's The Citizen edited by Dostoevsky in the detailed list that Rambaud provides of newspapers and journals being published in Russia.115 Nevertheless, after exploring the possible identity of the intended recipient of Dostoevsky's note, the French connection is being discounted for now, with Mackenzie Wallace becoming the more likely recipient.116 What is beyond dispute in our view is that Dostoevsky's autobiographical note was written with the intention of publication in a foreign newspaper for the consumption of readers abroad, who knew little about him; hence he introduces himself as a "Russian writer". On first reading it sounds very precise and formal, and reads almost like an official logbook or register of events presented from the third person point of view. It provides a chronological, authoritative-sounding introduction to his social background, life and works, opening with a few words on his place of birth, his father's social estate: "a noble, landowner and doctor of medicine", his early education in Moscow, followed by his passing an entry examination at the Main Engineering Academy in St Petersburg, and his later graduation: "In 1842 he completed a military engineering course and left the college as an engineer with the rank of sub-lieutenant and was retained on service in St Petersburg". At the time of writing the military was celebrated and a number of his Engineering Academy contemporaries had achieved distinction, and Dostoevsky had been originally a part of that group. The formal, positive tone with emphasis on his military qualifications and service is interrupted with the admission that "other goals and ambitions irresistibly attracted him. In particular, he began to take an interest in literature, philosophy and history." When taken together with his later statement about his major novels, one is struck that in his assessment of his writing, he is implying that his works should not be judged solely within the framework of realist fiction or belles-letters, as practised by contemporary realist writers. Could that be the reason why he gives weight to the fact that he took up writing professionally after resigning from service in 1844, but that it was not solely literature that attracted him, but also "philosophy and history"? He highlights three of his major novels: "In 1866, upon the death of his brother and the cessation of the journal Epoch (Epokha), which he published, Dostoevsky wrote the novel Crime and Punishment (Prestuplenie i nakazanie) and then, in 1868, the novel The Idiot (Idiot) followed by the novel Demons (Besy) in 1870. These three aspects of the sentence, his own attention was diverted away from something that he probably wished to forget, namely his own involvement within a subgroup of the Circle, whose aim was to set up an illegal underground printing press that he escaped being charged for (18, (193) (194) . Then follows a relatively long account of all the subsequent events connected with his sentence comprising well over a third of the entire autobiographical note. He refers in very positive, deferential terms to Tsar Nicholas I and to Alexander ii for the various allowances that they made in relation to his case. His underlying intention appears to have been to publicise an image of himself as that of a reformed political miscreant, who had renounced his former misguided socialist beliefs and youthful associations, with the inference that this had been recognised by the Sovereigns. The biographical note appears designed to serve as one more step in his efforts at having his name completely cleared and removed from the police surveillance files of the Third Section of His Imperial Majesty's Own Chancellery (Tretie Otdelenie or iii otdelenie sobstvennoi E.I.V. kantseliarii) (see section D below). Its aim was to express remorse for his beliefs and actions together with renunciation of his former convictions. Dostoevsky could not but sound positive and laudatory in relation to the Tsarist regime. He fully justifies his arrest in 1849 for participation in an anti-government political conspiracy. He focuses on and gives credit to Tsar Nicholas I for commuting the death sentence by firing squad to hard labour and exile, and at the completion of his four-year term in Siberia having his rights as citizen restored: "Similar pardons occurred now and again subsequently, but this was the first instance". Here he is indirectly, but clearly comparing his fate to that of the aristocratic Decembrists, most of whom had spent almost a lifetime in Siberia (until pardoned in the amnesty granted by Alexander ii on his accession), and whose fates had been mythologised in popular Russian culture and consciousness. There is a sense here that by putting himself into the same league as the martyr-like Decembrists, whose fates were known abroad and identified with Russia's harsh autocratic regime, Dostoevsky was participating in the creation of his own myth as that of a martyr. Yet, at the same time he proceeds to interpret and embellish on the feelings of the late Nicholas I towards himself, stating that "this (the pardon -I.Z.) was sanctioned by the will of the late emperor Nicholas I who took pity on Dostoevsky's youth and talent". Dostoevsky appears proud both of having had his civil rights restored and at his successful promotions in the military after he was despatched as a rank and file soldier to the Siberian line battalion no. 7 in the town of Semipalatinsk, where he was promoted to the rank of non-commissioned officer after a year and, with the accession of the reigning emperor Alexander ii to the throne, made a full officer. Dostoevsky draws to a close with an acknowledgement of his open allegiance to Slavophilism, with the avowal that his former socialist convictions had changed greatly. He does not expand on when or how his convictions changed, nor mention anything directly to do with religious beliefs. It is customary for commentators, while discussing Dostoevsky's "Conversion" in Siberia to cite in support of this "rebirth" his letter to N.D. Fonvizina of early 1854 interpreting it as Dostoevsky's confession of his allegiance to Christ (28 (1), . None the less, his autobiography overall, despite its tone, sounds convincingly remorseful and 'Christian' . As to his admission of being an "unreserved Slavophile" (otkrytyi slavianofil) by conviction, we shall only note here that the Slavophile sentiment, at its inception and even during later development, before it turned into a doctrine was not identified exclusively with faith and Orthodoxy, but rather with the historical development of Russia, its geography, ethnology and geo-politics (aspects also dealt with in Wallace's book on Russia). But Dostoevsky's other intention in this autobiographical note was to emphasise that he suffered from his ill health for much of his adult life. There is a sense that he feels almost apologetic that he did not publish any major works between 1844 and "his first novel" in 1861 following imprisonment in Siberia, The Humiliated and Injured (Unizhennye i oskorblennye). Poor Folk earned him a place in literature and was received extremely favourably by critics and leading lights in Russian society, being "a rare success in the full sense of the word. But the constant ill health which subsequently affected him for several years in a row impeded his literary activity." After Siberia: "As a victim of epilepsy, which he had acquired during his period of hard labour, he was discharged from the army in 1859 and returned to Russia". He underscores his best known work published after his "first novel": "But during the next two years he began and completed Notes from the House of the Dead (Zapiski iz mertvogo doma), in which he described his life of hard labour and wrote of his former fellow prisoners under fictitious names." Here he is emphasising the autobiographical elements of the book, rather than fictional ones. This is in contrast to his original intention when preparing it for publication to endow the narrative with elements of fiction, such as an invented narrator, whose crime was the murder of his wife. By identifying his creative work with his biography an artist participates in the creation of biographical myths and its diffusion into public consciousness. Dostoevsky is careful to note, in tune with the rest of his almost exalted assessment of the autocracy, that the practices and customs described in his account have long since undergone change in Russia. The emphasis on his "constant ill-health" in the early part of his career makes one wonder whether he is also implying that because of it he may have had less control over his physical and psychological condition, which may have been one reason why he might be excused in retrospect for becoming involved with the Petrashevsky Circle, and the associated complex personal relations that it spawned. Another reason could have been his ongoing desire to counteract rumours circulating about him about the earlier periods of his life, or even to forestall further rumours that he may have known were being spread privately and which he might have been expecting to emerge publicly in the future. In addition, he had been under the medical care of Dr S.D. Yanovsky and dependent on the latter's remedies (including opiates normally prescribed at the time), though his malady was never fully explained or diagnosed. It is significant that later he tried to let his relationship with the doctor become more distant. A similar progression (namely a withdrawal) occurred with his relationship with A.E. Wrangel, whom he also distanced himself from, although earlier Wrangel had zealously helped Dostoevsky in his tactics to attain a pardon, permission to publish his writing and reside in the capitals. Dostoevsky's description of himself being "a victim of epilepsy, which he had acquired during his period of hard labour" was also cited by him in official documents, when applying for a foreign passport for travel abroad, the official purpose always being to seek treatment for his medical condition. Dostoevsky's focus on his ill health throughout his adult life could have been due also to his realisation from the vantage point of his current age of around 56-57 years (and he was to die some three-four years later in January 1881), that his ongoing neuropsychological deterioration was manifesting itself unrelentingly. Today neuropsychological impairment is regarded as an unavoidable consequence of chronic epilepsy, predating the time it became active and manifested itself in actual epileptic seizures. It is now medically recognised that neoropsychological markers not only antedate the first seizure, but that these continue to influence cognition, and can manifest themselves in various psychiatric, behavioral and other developmental processes. Around the time when Dostoevsky was dictating his autobiographical note he may have been becoming more conscious of some of these processes, especially his increasing loss of memory and deterioration in recognising faces. In a letter dated 27 March, 1878 in reply to an unidentified person named Leonid Vasil'evich, who had written to Dostoevsky as someone, who had known the writer during his early St Petersburg years, Dostoevsky replies that he cannot remember him: "I must tell you that I suffer from epilepsy. Which deprives me completely of my memory, especially of certain events. I don't know if you will believe me, but I do not recognize people in the street to whom I was introduced only a month before. In addition, I completely forget my own works. This winter I reread one of my novels -Crime and Punishment -which I wrote ten years ago, and more than two thirds of the novel was completely unfamiliar to me, just as if I had not written it, to such an extent had I managed to forget it. " (31(1), 16-19) . Dostoevsky was about to embark on the composition of his next and last major novel Brothers Karamazov that he is likely to have recognised would be a massive task physically and his note was dictated before he was about to submerge himself in his new work. He sounds very proud as he sums up the success of his Writer's Diary for 1876-1877, yet he began producing it in 1873, as a column for the newspaper-journal The Citizen (Grazhdanin), when he was its editor, while the publisher was Prince V.P. Meshchersky. But he avoids any mention of his editorship of The Citizen, nor his association with Meshchersky, making it absolutely clear that he is the Diary's sole authorcreator: "In 1876 Dostoevsky began to publish a monthly journal in the original form of a "Diary", which was written by him alone, with no collaborators. This publication came out in 1876 and 1877 with a circulation of 8,000 copies. It was successful." Shortly before in the biographical entry about him in 'Silhouettes' of fifty Russian literati and their short biographies (1877) it was stated slightly ambiguously that "At the moment Dostoevsky is publishing a collection of articles on contemporary life, entitled Writer's Diary." [В настоящее время Ф.М. Достоевский издает сборник статей современной жизни, под названием «Дневник писателя»].119 Dostoevsky seems to want to ensure that Writer's Diary is seen as being exclusively his own work (not as a "collection of articles" that might have been implied). On initial reading, the account seems acceptable enough in relation to the known events in Dostoevsky's life, yet when one considers it more closely, the number of emphases and omissions it contains could possibly indicate that by the end of 1877 he had developed almost an instinctive or mechanical strategy regarding his self-representation to the public and to authorities. After the introduction to his early life, followed by a depiction of incidents that includes conflict that engages the reader, the rising action builds up to the essential message of the narrative, like an editorial, leading to the crunch-line: "Even from his literary opponents Dostoevsky has earned the reputation of being a highly honourable and sincere writer." His final crucial testimony was his admission that he was an "undisguised Slavophile" by conviction and that his former socialist beliefs had changed rather markedly (27, (120) (121) trips abroad, his experiences while living in different parts of Russia, his contribution to the literary organisations he was a member of etc. The focus is on St Petersburg, as if he wished to be associated only with the St Petersburg tradition in Russian culture, despite expressing allegiance to Slavophilism. Yet, looking back on his earlier activities and networks one notes that Dostoevsky did not express any adherence to Slavophilism prior to his imprisonment in the Petropavlovsky fortress in St Petersburg following his arrest in April 1849. On the contrary, his allegiances were with the Westerners and the more progressive literary groups in St Petersburg, some of whose members professed revolutionary sympathies. But when he was required to write a formal statement for the specially appointed Investigating commission to outline his contacts with and opinions of members of the Petrashevsky Circle, Dostoevsky tried to downplay his involvement with and the political importance of Petrashevsky by resorting to Slavophile-driven argumentation. The Investigative committee was anxious to be informed of the "threat to society" that the Circle posed, which Dostoevsky successfully eluded, by aligning himself with certain tendencies that were identified with nationalist, conservative Slavophile perceptions of the Russia state. While facing the accusation of practising "freethinking" (vol'nodumstvo), Dostoevsky tried to deflect this by questioning in a disarmingly innocent way, whether by talking about the crisis in the West it followed that he was a "freethinker" or of "republican persuasion"? How could anyone in Russia "even think about a Republic"? Western Europe and Russia had followed completely different historical paths of development: "Our country did not take shape along Western lines. We have historical examples in front of us."(18, 123)120 The "current crisis" in the West and the revolution there, Dostoevsky explained in his written statement could be attributed to "historical necessity" (istoricheskuiu neobkhodimost'): "There (i.e. in the West -I.Z.) for many centuries, for more than a millenium, a very persistent struggle has dragged on between society and the authority that has imposed itself onto an alien civilisation by means of conquest, violence and oppression (zavoevaniem, nasiliem, pritesneniem) ." (18, 123) When Dostoevsky returned to St Petersburg after his imprisonment and exile in Siberia, the public expression of his views from 1861 onwards on all trends in Russian society and culture were confined to the pages of the monthly journal Time (to be replaced later by the journal Epoch). Official permission from the St Petersburg Censorship Committee was given on the understanding that Time follow the official line or "trend" (napravlenie) that was set out in its original application. This was to polemicise with all literary factions, as its editors announced in several newspapers in 1860: to expose (oblichat'), or polemicise and denounce "all the literary oddities" (vse literaturnye strannosti), without taking obvious sides, though nevertheless promoting a specific ideology that was subsequently associated with 'pochvennichestvo ' (18, 39) . "We are not talking here about Slavophiles or Westernizers. Our time is completely indifferent to their domestic strife. We are talking about the reconciliation of civilization with the people's principle" (18, 37). Its various declarations about Slavophiles in the following issues were numerous and contradictory. Dostoevsky's most explicit declaration regarding Slavophilism was expressed in his Writer's Diary, July-August 1877 (25, (185) (186) (187) (188) (189) (190) (191) (192) (193) (194) (195) , approximately near the time when the autobiographical note was dictated. But perhaps some of the focus on Slavophilism was prompted by Dostoevsky's indistinct memory of the possible intended recipient of his note, Wallace, whom he had first met at Aksakov's some years earlier, and who was known to be fascinated by the phenomenon of Slavophilism, as indeed were many Russophile Britons. As Dostoevsky explains in his memorandum, after the expiry of his fouryear sentence of hard labour in the Omsk prison fortress, followed by service in the 7th Siberian line battalion, his civil rights, that he had lost in 1849 for participating in the Petrashevsky circle, were restored. The passport issued to him upon his discharge on June 30, 1859, in the town of Semipalatinsk, with permission to return to European Russia did not indicate that he would continue to be under police surveillance. Nevertheless, he and his wife believed that this surveillance was continuing. He states that this was demonstrated to him at the Third Section Of His Imperial Majesty's Own Chancery 126 to which he was always required to make a special request when departing abroad. In addition, while spending the winter of 1874-1875 in the town of Staraya Russa, his wife learnt from the local police superintendent that Dostoevsky continued to be under observation. At the time of writing of this appeal, Dostoevsky was convinced that the police surveillance had not been terminated. The appeal is dated in Dostoevsky's Complete Collected Works in 30 volumes as 30 March, 1879 (31(1), 246-27; 397-398) , although V.S. Nechaeva's dates it as 30 March, 1880, while G.F. Kogan is also inclined to date it as 1880.127 In his memorandum Dostoevsky assures the Minister of the Interior of his allegiance to the regime: "I have voiced and continue to voice my convictions, both political and religious, over hundreds of pages. These convictions, I hope, are of such a nature as not to give cause for suspicion of my political morality." (31(1), 246-247).
D However, researchers in Russia (specifically G. F. Kogan) have demonstrated that police surveillance on Dostoevsky had been lifted already in 1875.128 This occurred after a special commission was set up at the Ministry of the Interior to revise the rules on police supervision and administrative expulsion after it had received notifications from its various offices that too many cases had accumulated to monitor surveilled persons, complicating and burdening police and office work. The commission was instructed to consider in detail the reasons for having taken an "administrative measure" against each person, to collect certificates from the cases being carried out in different places and to trace the governors' attestations about them. The commission received from the office of the St. Petersburg mayor a list of persons who, having been in St. Petersburg under police supervision at different times since 1854, had not been involved in reprehensible acts and were presented for release from the police surveillance established for them.There were 43 such persons and Dostoevsky's name was amongst these. Detailed reports were provided of the cases in which his name had been mentioned in the past and the enquiries carried out about him, when he had applied for a passport for travel abroad. A note from the St Petersburg mayor was included stating that Dostoevsky had been tried in 1849 in the case of Butashevich-Petrashevsky, in 1856 he was pardoned. After that, he was repeatedly allowed to travel abroad, so he was recognized as being quite trustworthy and now there were no obstacles to releasing him from surveillance. The opinion about Dostoevsky's trustworthiness would have been formed on the recommendation of influencial persons from government circles to whom Dostoevsky had became close in the 1870s. But it took a very long time for that decision to be implemented, and for it to trickle down to the offices of governors and mayors, especially in the provinces, and for local administrators to put it into effect. By that time Dostoevsky was a resident at Staraya Russa. The decision by Dostoevsky to take some action eventually that resulted in his appeal was precipitated originally by some complications that arose, as he explains in his memorandum, when he needed to apply for a passport for foreign travel in April 1875. In her memoirs Anna Dostoevskaya relates that she was helping Dostoevsky to apply through the governor of Novgorod. She went to the local police officer to make some enquiries and during the course of their conversation he showed her a file: "the contents consisted of 'The Case of Retired Lt. Fyodor Mikhailovich Dostoevsky, under Secret Surveillance and Residing Temporarily in Staraya Russa'".129 She told this news to her husband, laughing at the absurdity of this fact, but he "took the news hard". They realised the reason why in the previous year the letters she was mailing from Staraya Russa to Dostoevsky in Bad Ems were held up and never arrived on time: "Our letters were being censored, and their mailing depended on the permission of Although the year 1875 is generally accepted today as being the official date when police surveillance on Dostoevsky was lifted, the fact remains that neither Dostoevsky, nor his wife believed that to be the case. She explains that Dostoevsky did not raise the issue of releasing him from police suveillance at the time, because some influential persons insisted that he would not have been allowed to be editor of a newspaper (The Citizen) and publisher of Writer's Diary unless the police surveillance had been lifted. She seems to have been the one particularly indignant at the police surveillance. According to her reminiscences, in 1880 "during the Pushkin memorial celebrations, Fyodor Mikhailovich had occasion to speak about it to a highly placed personage by whose order the secret surveillance was finally discontinued."131 That "highly placed personage" was Alexander Alekseevich Kireev (1833 Kireev ( -1910)) , a cavalry general and a prominent conservative Slavophile publicist, whom Dostoevsky approached. He was also the brother of Olga A. Kireeva-Novikova (mentioned in Section C above in connection with her having assisted Mackenzie Wallace with his book Russia). Kireev was an adjutant of the Grand Duke Konstantin Nikolayevich Romanov. As Secretary of the St Petersburg branch of the Society of Devotees of Spiritual Enlightenment, he and Dostoevsky would have had an opportunity to meet there initially. Anna Grigorievna may not be entirely correct about Dostoevsky first approaching Kireev at the Pushkin Festivities in Moscow in June 1880, where Dostoevsky delivered his famous Pushkin Speech on June 8. In accordance with a letter from Kireev to Dostoevsky, the researcher G. Kogan believes that the conversation between Dostoevsky and Kireev could have taken place as early as February or March 1880. On March 10 Kireev had written to Dostoevsky citing the Minister of the Interior, who had said that the removal of police supervision would not meet with any obstacles. Kireev repeated the latter's pronouncement to Dostoevsky that no one but Dostoevsky had the right to make any statements on his behalf and he asked Dostoevsky to deliver to him "for greater speed" his memorandum to the Minister. Kireev's letter to Dostoevsky does not indicate the year (only the day and month), and Anna Grigorievna has dated the letter by hand conditionally, indicating in brackets the year 1879.132 Dostoevsky's memorandum was apparently delivered to the offices of the Ministry of Internal Affairs (30(1), 397-398). It is characteristic of Dostoevsky to have approached an influential personage, such as Kireev directly in order to seek help. He did the same after his release from the Siberian prison when he approached General Eduard Totleben in 1856 to intercede on his behalf regarding his promotion to an officer's rank and for permission to be published.133 It is a reflection of how decisions were made in Russia at the highest level, where there were no clear mechanisms within the government for procedures to be followed to achieve justice. However, Kireev instructed him on the process to be followed, as prescribed by the Minister of the Interior, which resulted in the writing of the official memorandum. Kireev's impression of Dostoevsky and his favourable attitude towards the writer is reflected in a diary-entry that he wrote at the Pushkin Festivities in Moscow in 1880: "Turgenev is a perfect ramolli, does nasty things, allows all sorts of riff-raff (like the editorial staff of Golos) to exploit his name in its fight against Dostoevsky, about whom this party tells the devil knows what. Dostoevsky is a Christian and of a conservative trend of mind, and with his enormous talent and growing popularity among young people, he is dangerous to our nihilists in uniforms. Inde irae! <That's where the anger comes from! (lat.)> Turgenev goes to any lengths because of petty pride (but colossal in its pettiness). Quelle degringolade!.."134 From the time of Anna Grigorievna's encounter with the police officer (E.M. Gotskii-Danilovich) in Staraya Russa in the spring of 1875, and until the memorandum of 1880, Dostoevsky acted in accordance with his conviction that that he was still under police surveillance. The researchers Zhavoronkov and Belov also believe that the surveillance continued throughout the 1870s until the beginning of the 1880s, despite their publication of the contents of the file on Dostoevsky held in the archives of the Governor of the Novgorod region, that included item No. 50, dated 5 January 1876 where it stated that in accordance with the proposal of the regulatory superintendent of the Ministry of the Interior dated July 9 of the previous year Dostoevsky was being released from police surveillance. If one reads carefully Dostoevsky's letters to his wife 133 Military engineer E. I. Totleben (1818-1884) immortalized his name with the defense of Sevastopol. Dostoevsky met him in the 1840s and turned to him in 1856 with requests for intercession. Totleben took a lively part in his fate: "All my life I will remember his noble deed towards me. " (xxviii(1) 214-215; 225; 240-241; 342-343; 371-372) 134 Quoted in Aksakov Ivan Sergeevich. Materialy dlia letopisi zhizni i tvorchestva. Vypusk 6: v dvukh chastiakh. 1880 -1886 . Redaktor-izdatel' gazety "Rus'", chast' 1: 1880 -1884 in Staraya Russa written from abroad, or even from the capitals, one can notice a slight change of tone to that of the pre-1875 letters. When examined through the prism of Dostoevsky's conviction that they were being read by officials, one senses that at times Dostoevsky may have deliberately written in accordance with the expectations that he thought they might have had of private letters to one's wife, and even possibly attempted to liven up provocatively their reading. Nor did he hide from them the fact that he knew the letters were being perlustrated (29(2), 51). Some of the earlier letters have survived only in copies that had been "censored" by Anna Grigorievna by having lines crossed out or erased,135 and on following the letter transcribed (31(1), . No doubt, had there been something that she herself would not have wanted to be read in her own correspondence (and not only to her husband), she would have taken appropriate precautions. ii, to the throne, made a full officer. As a victim of epilepsy, which he had acquired during his period of hard labour, he was discharged from the army in 1859 and returned to Russia -first to the city of Tver' and then to St Petersburg. Here Dostoevsky took up literature once again.
Bibliography (Russian) In 1861 his elder brother, Mikhail Mikhailovich Dostoevsky, began to put out a large literary monthly with the title of Time (Vremia). F. M. Dostoevsky also contributed to the publication of the journal and in it he published his first novel The Humiliated and Injured (Unizhennye i oskorblennye) which was sympathetically received by the public. But during the next two years he began and completed Notes from the House of the Dead (Zapiski iz mertvogo doma), in which he described his life of hard labour and wrote of his former fellow prisoners under fictitious names. This book was read right throughout Russia and is still highly esteemed, even though the practices and customs described in Zapiski iz mertvogo doma have long since undergone change in Russia. In 1866, upon the death of his brother and the cessation of the journal Epokha, which he published, Dostoevsky wrote the novel Crime and Punishment (Prestuplenie i nakazanie) and then, in 1868, the novel The Idiot (Idiot), followed by the novel Demons (Besy) in 1870. These three novels were highly regarded by the public, even though Dostoevsky may have given through them a very harsh picture of contemporary Russian society. In 1876 Dostoevsky began to publish a monthly journal in the original form of a "Diary", which was written by him alone, with no collaborators. This publication came out in 1876 and 1877 with a circulation of 8,000 copies. It was successful. In general Dostoevsky is popular with the Russian public. Even from his literary opponents Dostoevsky has earned the reputation of being a highly honourable and sincere writer. He is an undisguised Slavophile by conviction; his former socialist convictions have radically changed. nevertheless, this surveillance is continuing, as, for instance, was communicated to me at the Third Section of His Imperial Majesty's Own Chancellery, to which I was always required to make a special request when departing abroad, and finally, back in 1875, when, spending the winter of 1874-1875 in the town of Staraya Russa, I learned from the Staraya Russa police superintendent that he had me under observation.
D. Appeal to Since the time of my pardoning and the restoration of my civil rights some 25 years have passed. I have voiced and continue to voice my convictions, both political and religious, over hundreds of pages. These convictions, I hope, are of such a nature as not to give cause for suspicion of my political morality, and therefore I am taking the liberty of requesting that police surveillance over me be terminated. To 40 figure 1 Photocopy of document of the original layout of the graves of Maria Fedorovna and the Gardners, including Ekaterina Alfonsky at Lazarev cemetery: PAVEL E. FOKIN, 'O VOSSTANOVLENII PAMIATNIKA MATERI F.M. DOSTOEVSKOGO NA BYVSHEM LAZAREVSKOM KLADBISHCHE V MOSKVE' , NEIZVESTNYI DOSTOEVSKII (THE UNKNOWN DOSTOEVSKY: ONLINE RESEARCH JOURNAL), 1, 2017, 154.
figure 2 2 figure 2 The name of Alexander Gardner listed in the document of students studying at the Main Engineering Academy in 1841, at the time when F.M. Dostoevsky was also enrolled at the Engineering Academy. photocopy of page 98 from prilozhenie: [maksimovskii m.] istoricheskii ocherk razvitiia glavnogo inzhenernogo uchilishcha 1819-1869. sost. pri nikolaevskoi inzhenernoi akademii m. maksimovskim (spb.: tipografiia imperatorskoi akademii nauk, 1869). prilozhenie. 98.
69 http://www.hram-ks.ru/history.shtml 70 G.A. Fedorov, Moskovskii mir Dostoevskogo. Iz istoriirusskoi khudozhestvennoi kul'tury xx veka, (M.: Iazyki slavianskoi kul'tury, 2004) 48. (Unfortunately, Fedorov eschews detailed footnotes and does not provide a source for his quote from Kh.I. Oppel' . According to available information in Encyclopedias his dates are 1768-1835 and he was the chief physician at the Mariinsky Hospital from 1803 to 1830. Nor is there anything substantial about Alfonsky's private life in the hagiographic official-sounding article: V.K. Gostivshchev, M.A. Evseev, 'Zaveduiushchii kafedroi obshchei khirurgii, dekan meditsinskogo fakul'teta, prorektor, rektor Imperatorskogo Moskovskogo universiteta Arkadii Alekseevich Al'fonskii i ego rol' v sovershenstvovanii prepodavanii klinicheskikh distsiplin i razvitii meditsinskogo fakul'teta' , Sechenovskii vestnik. 2013. No 2(12) 5-9. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/Alfonsky acquired a second family when he remarried in 1830 the well-off Ekaterina Alexandrovna Mukhanova (1800-1876),71 daughter of the Ryazan governor, and from 1831 to 1841 they had six children of their own, while Ekaterina's children became Mukhanova's step-children. The reference to stepmother (machekha) interwoven with references to Alfonsky in Dostoevsky's Life of a Great Sinner may echo the lives of Ekaterina's sons, especially the eldest Arkady (the theme of step-childhood subsequently so important in The Adolescent). Fedorov also hypothesises that Aleksei Alfonsky, who was from the end of 1869 Assistant Chief of Police of the Moscow District could have supplied Dostoevsky with some information about the murder of Ivanov by the Nechaevtsy to be used in Demons. (Aleksey Alfonsky, b.1824, was following in the footsteps of his cousin Vladimir Gardner b.1819, who had served earlier as staff-captain in the Moscow Gendarme Division.)
76 F.M. Dostoevsky, Winter Notes on Summer Impressions. Translated with an Introduction by Kyril Fitzlyon (London: 1955) 2. 77 Ibid. A. Dostoevskii, 70.supersedes and corrects all previous accounts, such as: Ibid., A. Dostoevskii, 64-66; Ibid., Biblioteka; Ibid., Grossman, 68; Irene Zohrab, 'Dostoevsky and Kierkegaard in the context of State censorship' , The Dostoevsky Journal: A Comparative Literature Review, vols. 14-15: (2013-2014) 65. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
80 A. Dostoevskii, 57-58. 81 Ibid., A. Dostoevskii, 58. 82 F.M. Dostoevskii v vospominaniiakh sovremennikov, vols.1-2, (Moscow: Khud.Lit., 1964), I, 128. 83 Anna Dostoevsky, Dostoevsky. Reminiscences (London: Wildwood House, 1975), 17. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
90 Dubnensko-Taldomskoe blagochinie Sergievo-Posadskoi eparkhii Mck Mirop rpt s: https://www.facebook.com/dubnablago/posts/1687621238056552 91 'Kratkie biograficheskie svedeniia o F.M. Dostoevskom', F.M. Dostoevskii. Polnoe sobranie sochnenii. (SPb.: 1906) i-iii. 92 O.F. Miller, 'Materialy dlia zhizneopisaniia F.M. Dostoevskogo' , F.M. Dostoevskii. Polnoe sobranie sochnenii. (SPb.:1882-1883) 3. 93 Russkii entsiklopedicheskii slovar' Red. I.N. Berezin. (St Peterburg: 1874-1875), Otd. ii., T.I, 475. 94 F.M. Dostoevskii. "Odno slovo po povodu moei biografii", F.M. Dostoevskii. Polnoe sobranie sochnenii v tridtsati tomakh.
part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
107 A. Maikov, "Neskol'ko slov o F.M. Dostoevskom. (Chitano v publichnom zasedanii Peterburgskogo Slavianskogo Obshchestva 14 fevralia), Rus', No 18, 1881. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ of the material in the later 1912 edition.109 The 1877 edition was translated into Russian in 1880-1881.110
116 I wish to thank Dr Hilary Chapman for her help with checking Revue des Deux Mondes for the relevant years for articles relating to Russian literature. à l'Université pour devenir la propriété de M. Katkof. Parmi les revues présentant un intérêt général, figurent le Messager d'Europe, de M. Stasioulévitch, le Messager russe, de M. Katkof, le Citoyen, les Annales de la patrie, le Diélo (l'Action), organe avancé. D'autres ont un caractère spécialement historique: telles sont l' Archive russe de M. Barténief, l'Antiquité russe, la Russie ancienne et nouvelle, le Recueil de la Société impériale d'histoire russe, fondé en 1867, etc. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Interior Minister to have police surveillance lifted. memorandum to l. S. makov, minister of internal affairs March, 1979, St Petersburg Memorandum from Second Lieutenant (retired) Fedor Mikhailovich Dostoevsky. By virtue of my most gracious promotion to ensign in 1858 from the non-commissioned ranks in the 7th Siberian line battalion, which I joined upon expiry of my four-year sentence to hard labour (second grade) in the Omsk fortress, all my civil rights, which I lost for participation in criminal propaganda in St Petersburg in 1849, were restored. The passport issued to me upon my discharge on June 30, 1859, in the town of Semipalatinsk, does not indicate that I would be under police surveillance; part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
19 B Dostoevsky's Mother's Best Friend Ekaterina Alfonsky with British "Gardner" Connection? the writer's family "can testify to his care." Spirit (near the former graves of Maria and her friend), a church familiar to the Dostoevskys, a speaker (head of Museum-apartment of F.M. Dostoevsky) The neglected information to be considered, with its entangled connec- tions and intimations, is contained in some revealing notes in the Memoirs of Dostoevsky's younger brother Andrei Mikhailovich (1825-1897) relating to their mother, Maria Fedorovna Dostoevsky, who was born 14 Jan (26 Jan) 1800, and passed away on 27 Feb O.S. (11 March) 1837, when Dostoevsky had not yet turned 16.20 Her influence on Dostoevsky has been the subject of some interest, extending to the belief by some commentators that certain features of her per- sonality are reflected in the characterisations of the mother of Arkady in The Adolescent (Podrostok) and the mother of Alesha in The Brothers Karamazov (Brat'ia Karamazovy).21 She is also credited with "having taken a great interest" in the reading material of her children.22 Recently the tombstone on Maria Dostoevskaya's grave, originally at Lazarev cemetery destroyed in the 1930s has been restored at a grand ceremony in Moscow on March 11, 2017 on the territory of the former Lazarev cemetery, with the launching of an attendant exhibition.23 On this occasion, near the Church of the Descent of the Holy
taught English at Moscow University from 1828 onwards. He is the author of an anthology of English literature.58 54 Nikolai's son Evgeny married the writer Raisa Alexandrovna Koreneva, who was brought up together with D.I. Pisarev, the future critic, at the home of his mother. Pisarev loved Raisa and hoped that she would marry him, but she married into the Gardner clan. 55 Rozina, 192. 56 G.A. Fedorov, Moskovskii mir Dostoevskogo. Iz istoriirusskoi khudozhestvennoi kul'tury xx veka, (M.:Iazyki slavianskoi kul'tury, 2004) 112. 57 Fedorov, 114. 58 Edward Harvey was the compiler of The English Student's Manual or Miscellaneous pieces selected from the best English Language prose writers, preceded by a sketch of the rise and progress of the English Language, and followed by a copious English-French and Russian Dictionary of all the words contained in the body of the works, Moscow, 1835, 2 v. See Biograficheskii slovar' professorov i prepodavatelei Moskovskogo universiteta, ch.1, Moscow, 1855, 175-177. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
monuments next to it: "Alfonskaya Ekaterina Kirillovna", born Andreevskaya; Anna Gardner, born Andreevskaya; Vladimir Petrovich Gardner 1819-1862."63 She continues with the observations that the last named one, Vladimir Gardner, was the same age as the brothers Dostoevsky and could have been known to them in Moscow, and studied with them at the Engineering Academy.64 59 Literaturnoe nasledstvo. Russko-angliiskie literaturnye sviazi (xviii vek -pervaia polovina xix veka), 91, (Nauka: Moscow, 1982), 520, 528-29, 566-7. 60 Frank, The Seeds of Revolt; Joseph Frank, Dostoevsky. The Miraculous Years, 1865-1871, (USA: Princeton University Press, 1995). 61 Thomas Gaiton Marullo, Fyodor Dostoevsky-In the Beginning (1821-1845) A Life in Letters, Memoirs, and Criticism, (2017). 62 N. S. Nechaeva Rannii Dostoevskii. 1821-1849 (M.: 1979) 60-61. See also Letopis' zhizni i tvorchestva F.M. Dostoevskogo.V trekh tomakh 1821-1881, vol. 1 (Sankt Peterburg: Akademicheskii proekt, 1993) 40. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
2 January 1819 d. 8 January 1882; with Ek. Kiril. Alfonsky (Lazarevskoe cemetery)."66 Why is Ekaterina identified in the Nekropolis as being buried with Vladimir Gardner? Why is he not identified as being buried with his own mother Anna?67 Apart from Ekaterina there are no other Alfonsky family members listed as being buried at Lazarevo cemetery. Her husband Arkady (and his second wife, neé Mukhanova) were buried at Vagan'kovo cemetery.68 Ekaterina Alfonskaya's sons Arkady and Aleksey, and her daughter Anna were all buried (in 1890, 1872, 1888 respectively) at the more 63 Yet Vladimir's date of death is cited by other commentators as being 1882, unless it is a copying mistake in the digitalised version of Nechaeva. 64 Ibid. Nechaeva, 277-278. 65 Ibid. Fokin, 154. 66 Nikolai Mikhailovich, Velikii Knyaz' . Moskovskii nekropol': v 3 tomakh (St Petersburg: Tipografiia M.M. Stasiulevicha 1907), vol. 1. (A-I), 519 pp. Vol. I, 255. V.P. Gardner is identified here of having died in 1882, while according to Nechaeva's copied version he died in 1862. Why the discrepancy? Also why is he buried together with Ekaterina Alfonsky, friend of Dostoevsky's mother? 67 Ibid. 255. Also listed are: "Gardner, Konstantin Petrovich d. 11 March 1898 at 64 yrs; with S.P. and P.P. Gardner (Lazarevskoe cemetery)./ Gardner, Pavel Petrovich; without dates; with S. P. Gardner (Lazarevskoe cemetery)./ Gardner, Sergei Petrovich d. 28 September 1874; with K.P. Gardner (Lazarevskoe cemetery)." 68 Ibid., 29. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Dostoevskom)91 and it has remained unpublished in its entirety in English translation, until the current version in Appendix C. As was noted at the time of its first publication, the biographical note was dictated by Dostoevsky to his wife, Anna Grigorievna Dostoevskaya, who took it down in short Dostoevsky's Autobiographical Note Intended for Foreign Correspondent? Dostoevsky's attempt at writing a short autobiographical note that covers his life up to and including 1877 remains the only source of information composed entirely by him (27, 120-121). It was first published in Russia in 1906, titled "Short biographical information about F.M. Dostoevsky" (Kratkie biografich- eskie svedeniia o F.M.
. See: Sovremennye russkie deiiateli. Sbornik portretov zamechatel'nykh lits nastoiashchego vremeni s biograficheskimi ocherkami. t.ii. Ed. A. O. Bauman (SPb.: Tip. N.A. Lebedeva, 1877). 97 The autobiographical note was subsequently decrypted by A.G. Dostoevkaya for the 1906 edition of Dostoevsky's works. Later it was decrypted again by Ts. M Poshemanskaia for publication in Literaturnyi arkhiv in 1961. 98 Many British periodicals were also designated as 'Reviews' , including The Edinburgh Review and The Fortnightly Review to which K.P. Pobedonostsev subscribed. There was also Quarterly Review and Saturday Review to which Prince V.P. Meshchersky subscribed for the offices of The Citizen, when Dostoevsky was its editor. Other popular journals included The Contemporary Review. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
. Dostoevsky does not mention anything about his private life, his family, his interests, nor his professional associations etc. Though proud of the success of Writer's Diary, there is no reference to his other journalistic and publicist work, his frequent Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ part iii: New perspectives on Dostoevsky 119 "Siluety" piatidesiati russkikh literatorov, 50. The Dostoevsky Journal 23 (2022) 111-172
121 Dostoevsky superimposes onto Russian history an ideological interpretation that has similarities with one expressed by the Slavophiles, which demonstrates that Dostoevsky was familiar with various trends of political thought reflected in the Russian press of the time, including the theoretical debates between the Slavophiles and the Westerners. Comparable formulations can be found in a number of articles that had been published in the Slavophile journal Muscovite (Moskvitianin) in the 1840s.122 120 Ibid. 'Ob"iasnenie F.M. Dostoevskogo ' , pss, 18, 117-134, 123 . See also Translation into English in Dostoevsky as Reformer: The Petrashevsky Circle. Edited and translated by Liza Knapp. (Ann Arbor: Ardis, 1987), 33-34. 121 Ibid. Knapp, 33. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Appeal to Interior Minister to have Police Surveillance Lifted. Dostoevsky's memorandum addressed to the Minister of the Interior (who was L.S. Makov at the time), requesting that police surveillance of him be removed has had little exposure, and has never been translated in its entirety into English (until the current translation featured in the Appendix.)123 It has not been included into the five-volume collection of Dostoevsky's letters in English translation edited by David Lowe124. The researcher V. Nechaeva first publishedDostoevsky's original draft manuscript in 1934.125 122 For example in contributions such as For Native Antiquity (Za rodnuiu starinu) (1845), which was unsigned, but whose author was in fact M.P. Pogodin (since he republished this article in a two-volume collection Istoriko-kriticheskie otryvki (M.: 1846).). 123 'Dokladnaia zapiska ministru vnutrennikh del L.S. Makovu' , pss, 30(1), 246. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
police inspector".130 Anna's reminiscences were composed between 1911 and 1916 and published in 1925, so her assessments were made with the benefit of over 35 years of contemplation regarding the narrative's standpoint. 129 Anna Dostoevsky, 249 . See also: A. Zhavoronkov, S. Belov, 'Delo ob otstavnom podporuchike Fedore Dostoevskom' , Russkaia literatura, 4 (1963): 197-202. Here 17 documents from the police file on Dostoevsky are reproduced. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ a
Аксаков, Иван Сергеевич, Материалы для летописи жизни и творчества. Выпуск 6: в двух частях. 1880-1886 гг. Редактор-издатель газеты «Русь», часть 1: 1880-1884 (Уфа: УИКул МВД России, 2015). Андрианова И.С., «Письма Достоевского к жене: проблема цензурных исправлений А.Г. Достоевской». Неизвестный Достоевский, No 1/2 (2014): 65-71. Арбат, И.Ю., Фарфоровый городок (Москва: Московский рабочий, 1957). Бак, Д.П., Захаров В.Н., Погомарева Г. Б., Фокин П.Е., «Из выступлений участников пресс-конференции, посвященной восстановлению надгробного памятника на могиле матери Ф.М. Достоевского и прошедшей 9 марта 2017 года в Музееквартире писателя в Москве». Неизвестный Достоевский, No1 (2017): 160-165. Белов, С.В., Энциклопедический словарь Ф.М. Достоевский и его окружение в 2 т. (СПб: Алетейя, 2001). «Биография, письма, заметки», Ф. М. Достоевский, Полное собрание сочинений, Т. 1. (СПб.: Тип. бр. Пантелеевых,1883). Библиотека Ф.М. Достоевского. Опыт реконструкции. Научное описание. От. ред. Н.Ф. Буданова (СПб: Наука, 2005). Биографический словарь профессоров и преподавателей Московского университета, ч.1. (М., Унив. Тип., 1855). Бойчак, М., История Киевского военного госпиталя. Киевский военный госпиталь в xviii-xix веках. Становление и развитие военной медицыны в Украине (Киев: Пресса Украины, 2005), Кн. 52. 135 I.S. Andrianova, "Pis'ma Dostoevskogo k zhene: problema tsenzurnykh ispravlenii", Neizvestnyi Dostoevsky, No 1/2 (2014): 65-71).
His Excellency Minister of Internal Affairs. March, 1879. Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ zohrab The Dostoevsky Journal 23 (2022) 111-172
Irene Zohrab, 2022 | doi:10.1163/23752122-02301012 The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
V.A. Fateev, 'Trezvyi sredi prorochestvuiushchikh, ili vsegda li
x 2=4?' , N.N. Strakhov: Lichnost' . Tvorchestvo. Epokha (Sankt-Peterburg: 'Pushkinskii Dom' , 2021) 279-294; N. N. Strakhov, 'Nabliudeniia. (Posv<iashchaetsia> F.M. D<ostoevskomu>' , F.M. Dostoevskii. Novye materialy i issledovaniia. Literaturnoe nasledstvo, 86 (M. Nauka, 1973); L. M. Rosenblium, 'Tvorcheskie dnevniki Dostoevskogo' , Neizdannyi Dostoevskjii. Literaturnoe nasledstvo, 83. (Moscow: Nauka, 1971): 17-19; N.N. Strakhov, 'Biografiia, pis'ma, zametki' , F.M. Dostoevskii, part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
V.P. Meshcherskii Pis'ma k velikomu kniaziu Aleksandru Aleksandrovichu, 1869 -1878. Chernikova N.V. (predislovie, kommentarii). (M.: Novoe literaturnoe obozrenie, 2014) 419.
"Spisok nesokhranivshikhsia i nenaidennykh pisem i delovykh bumag 1869-1874", F. M. Dostoevskii, Polnoe sobranie sochinenii v tridtsati tomakh (Leningrad: Nauka, 1972-90), 29(1), 551-562. Hereafter references to this edition pss will be indicated within the text as bracketed volume and page numbers.Polnoe sobranie sochinenii, t.1. (SPb.:1883) 244; V.N. Zakharov, 'Skol'ko budet dvazhdy dva, ili neochevidnost' ochevidnogo v poetike Dostoevskogo' . Voprosy Filosofii. No
(2011): 109-114. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Irene Zohrab, 'Correspondence: Grand Duke Nikolai Alexandrovich Romanov and Vladimir Petrovich Meshchersky' , New Zealand Slavonic Journal. Slavonic Journeys Across Two Hemispheres: Festschrift in honour of Arnold McMillin (Wellington: vuw, 2003), 237-280.
Chernikova, 419.part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Chernikova, 419. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Fyodor Dostoyevsky. Complete Letters: Volume Four. 1872 -1877. Edited and translated by David A Lowe, (Ann Arbor: Ardis, 1991), 58-59.
Grossman,
part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Complete Letters.Vol 4,
16 AnnaDostoevsky, Reminiscences, (London: Wildwood House, 1975) 181. 17Ibid. 317. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Memuary grafa S.D. Sheremet'eva, prim. L.I. Shokhina (M.: "Indrik",
2001) 572. 20 A.M. Dostoevskii, Vospominaiia (Moscow: Agraf, 1999): 33. The first edition of A. Dostoevsky's Reminsicences appeared posthumously in 1930, edited by the author's son,
Letopis' zhizni i tvorchestva F.M. Dostoevskogo.V trekh tomakh 1821 -1881 , vol. 1 [Sankt Peterburg: Akademicheskii proekt, 1993) 19.
Belov, See also: V.K. Gostivshchev, M.A. Evseev, «Zaveduiushchii kafedroi obshchei khirurgii, dekan meditsinskogo fakul'teta, prorektor, rektor Imperatorskogo Moskovskogo universiteta Arkadii Alekseevich Al'fonskii i ego rol' v sovershenstvovanii prepodavanii klinicheskikh distsiplin i razvitii meditsinskogo fakul'teta», Sechenovskii vestnik. 2013. No 2(12) 5-9. part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
K.A. Barsht, Tekstologicheskoe issledovaniie zapisnykh tetradei F.M. Dostoevskogo k romanu "Besy". Diplomaticheskaia transkriptsiia,(S-Pb: Nauka, 2021). 48, 102, 104, 108. 110-113, 373, 424.
Anthony Cross, By the Banks of the Neva: Chapters from the Lives and Careers of the British in Eighteenth century Russia, Cambridge University Press, 1997, 73. See also Gardner's Memorial plaque at Verbilki: https://web.archive.org/web/20100918013307/http:// poselokverbilki.ru/verbp25.htmlSee also: https://www.biografija.ru/biography/gardnerfranc-yakovlevich.htm
30 There has been some uncertainty as to whether Gardner was an Englishman from Stafforshire or a Scotsman.There are various different transliterations of his name and patronymics of his children. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
William Tooke, View of the Russian Empire during the Reign of Catharine the Second and to the Close of the Present Century, iii (London, 1799), 505.
Cross, By the Banks of the Neva, 73; Ibid., William Tooke; I.Iu. Arbat, Farforovyi gorodok, Moscow, 1957; Marvin C. Ross, Russian Porcelains, (University of Oklahoma Press, 1968).
Cross, By the Banks of the Neva, 73; 27,
O.V. Rozina, 'Farforovaia fabrika F. Ia. Gardnera i ego potomkov: istorikokul'turologicheskoe issledovaniia' , Verkhnevolzhskii filologichaskii vestnik (2019): 188-196; V.P. Kuz'mina, 'Farforovyi zavod Gardnera. Manyfaktury Gardner v Verbilkakh' , Rossiia. Razvitie tekhnologii polucheniia farfora, 2021, 1-75.part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Anglofiliia u trona. Britantsy i russkie v vek Ekateriny ii: Katalog vystavki. Sost. Anthony GlennCross (British Council, 1992).
V.P. Kuz'mina, 'Farforovyi zavod Gardnera' , Razvitie tekhnologii polucheniia farfora, 2021; N.A. Kruglianskaia, 'Gardnera farfor' , Universal'naia entsiklopedia Kirilla i Mefodiia. https:// megabook.ru ›
Geni World Family Tree.
M.P. Boichak, Istoriia Kievskogo voennogo gospitalia. Kievskii voennyi gospital' v xviii-xix vekakh. Stanovlenie i razvitie voennoi meditsiny v Ukraine, (Kiev: "Pressa Ukrainy", 2006), Kn. 52, 140-143.
According to O.V. Rozina, 'Farforovaia fabrika F.Ia. Gardnera i ego potomkov: istorikokul'turologicheskoe issledovaniia' Peter Frantsevich (also spelled Francevic) Gardner was the son of the founder F.Ia. Gardner. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
A.I. Savel'ev, 'Vospominaniia o F.M. Dostoevskom' , F.M. Dostoevskii v vospominaniiakh sovremennikov, (M.:Khudozh. Lit., 1964) 96-105.
Joseph Frank, Dostoevsky. The Seeds of Revolt, 1821-1849, (USA: Princeton University Press, 1976) 80.
Belov, F.M., Dostoevskii i ego okruzhenie, ii, 75;
The Geni site is managed by a "Markova Irina". According to the Rodovod site (that has some discrepencies) nine sons of Anna Gardner are listed. Rozina provides similar information.
Shkoly voennykh inzhenerov v 1701Shkoly voennykh inzhenerov v -1960 godakh: https://viupetra2.3dn.ru/Istoriia, kultura i traditsiia Riazanskogo kraia.part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
She was sister of the Decembrist Petr Alexandrovich Mukhanov. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Nechaeva, 8.
V.N. Zakharov, 'Pamiatnik kak tekst, ili poslanie brat'ev Dostoevskikh' , NeizvestnyiDostoevsky, 1, 2004: 5-11.
Biblioteka F.M. Dostoevskogo (Sankt-Peterburg: Nauka, 2005); L.P. Grossman Seminarii po Dostoevskomu.(Moskva, Petrograd: Gos. Izd., 1922).
The recent research by B.N. and N.A. Tikhomirov '"Sto chetyre sviashchennye istorii " I. Gibnera v zhizni i tvorcheskoi rabote Dostoevskogo' in Ibid., Novye arkhivnya i pechatnye istochniki, 27-60 (preceded by their 'Tak kakoe zhe izdania knigi I. Gibnera mog chitat' v detstve Dostoevskii?' , Dostoevskii i mirovaia kul'tura. Al'manakh No 36, (2018): 19-31) part iii: New perspectives on Dostoevsky The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
Ibid. 'Otchet o Venskoi Vystavke' .
Ibid. 729. zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
zohrab The Dostoevsky Journal 23 (2022) 111-172 Downloaded from Brill.com 03/23/2024 10:59:22AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/
|
10.5713/ajas.18.0302
|
cc-by
| null | null |
openalex
|
Objective: In the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis. Methods: We conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: Through RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle. Conclusion: These results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.INTRODUCTION The most important economic traits in poultry industry are egg production and amount of meat. Thus, genetic selection and breeding programs have made efforts to improve such quantitative traits . Particularly, chicken meat yield is closely related to skeletal muscle growth and so, the understanding of muscle proliferation and differentiation could provide the critical tool for improving the meat amount. Muscle growth and differentiation in vertebrates including Aves are controlled by a complicated processes. For in vitro analysis of muscle growth, the cell culture systems of muscleoriginated cells have been established in various species . Many studies on myoblasts which are the precursors of myotubes were conducted to examine the regulatory pathways for muscle differentiation . In Aves, QM7 cell line derived from quail myoblast has been utilized for studying the myogenesis mechanisms [4, 5] . In mammals, the onsets of myopathy were caused by the dysregulated myogenic factors such as myogenic regulatory factors (MRFs) . MRFs include a group of four proteins; MyoD, Myf5, Myogenin, and MRF4 [7, 8] . Those MRFs regulate cell cycle arrest of precursor muscle cells and regenerative proliferation. Additionally, MRFs induce muscle differentiation and sarcomere assembly process by activating sarcomere and expressing muscle specific genes . Calcium ion is well known to have various biological functions in body as formation of bone structure, wound healing, and hormone seduction . In muscle, calcium ion acts as a regulatory molecule and signaling molecule in muscle fiber . Generally, calcium signal in muscle is closely related to muscle contraction and muscle relaxation . The muscle contraction pathways regulated by calcium ions show three major mechanisms ; i) Troponin-tropomyosin complex system which is related to actin-filament in skeletal muscle and cardiac muscle, ii) Myosin light chain kinase mechanism which occurs during muscle contraction with calmodulin in vertebrate's smooth muscles, iii) Calcium ions bind directly to myosin and induce muscle contraction. Thus, calcium ions are very important for muscle contraction and relaxation but the roles of calcium ions during muscle differentiation have not yet been studied. Based on mRNA sequencing analysis of quail myoblasts (QM7) during 3 days of myotube differentiation, we analyzed the differentially expressed genes (DEGs) and validated the expression profiles of calcium-related genes to investigate calcium regulatory processes during myotube differentiation.
MATERIALS AND METHODS
Quail myoblast culture According to the previous reports [4, 5] , QM7 cells (American Type Culture Collection, Manassas, VA, USA) were maintained at 37C in an atmosphere of 5% CO 2 and 60% to 70% relative humidity with Medium 199 containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA), and 1× antibioticantimycotic (Invitrogen, USA) by subculturing the cells at 70% confluency. To induce myotube differentiation at 90% confluency, the differentiation medium containing 0.5% FBS and 1×antibiotic-antimycotic was changed and half of the medium was replaced daily with fresh differentiation medium.
Assembly of RNA-sequencing data of quail transcripts Total mRNAs were isolated from the differentiated QM7 cells during 3 days of myotube differentiation as well as the undifferentiated QM7 cells, and all of samples were triplicated. We generated RNA-sequence data in the QM7 cells. Sequencing of an RNA-sequencing library for each sample was carried out using Illumina HiSeq2500 (Illumina, San Diego, CA, USA) in order to generate 100 pair-end reads. These sequences were aligned and mapped against the chicken reference genome using TopHat for paired-end sequences.
Total RNA isolation cDNA synthesis To validate the expression patterns of calcium-related genes, total mRNAs were isolated from the undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). The harvested cells were dissolved using 400 μL of TRIzol (Invitrogen, USA) and 100 μL of chloroform was added to remove the organic solvent. After final washing with 75% ethanol, mRNA pellets were dissolved in RNase-free water. The extracted mRNAs were confirmed by measuring the absorbance at 230 nm and 260 nm using a spectrophotometer (NanoDrop 2000, Thermo Scientific, DE, USA) and stored at -70C for the next experiments. To synthesize cDNA, 2 μg of RNA, 1 μL of oligo-dT (Invitrogen, USA), and 1 μL of RNase-free water were added and then the mixture was denatured at 80C for 3 min. Subsequently, the cDNA was synthesized using 4 μL of 5× RT buffer, 5 μL of 2 mM deoxynucleotide (dNTP), 0.5 μL of RNase inhibitor (Promega Corporation, Madison, WI, USA) and 1 μL of Moloney Murine Leukemia Virus reverse transcriptase (Promega, USA).
Polymerase chain reaction and quantitative polymerase chain reaction amplification The polymerase chain reaction (PCR) reactions to amplify the target genes in the cDNA were carried out under the following conditions; 1.8 μL dNTP, 2 μL 10× buffer, 0.2 μL Taq, and 12 μL distilled water were added to 2 μL of 50 ng/μL diluted cDNA, and 5 pmol/μL diluted forward primer and reverse primer. The PCR was carried out in a total volume of 20 μL and the PCR procedure was; denaturation at 94C for 10 min, and a second denaturation at 94C for 30 s, followed by annealing at 55C for 30 s and extension at 72C for 30 s. This step was repeated for 38 cycles and then a final extension was performed at 72C for 10 min. The band was confirmed on UV light using a 1.5% SeaKem LE agarose gel (Lonza, Rockland, MD, USA). For quantification of calcium-related genes, quantitative reverse transcription-PCR (qRT-PCR) analysis was performed using the iCycler iQ Real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and EvaGreen (Biotium, Fremont, CA, USA). qRT-PCR data of the target genes were normalized relative to beta-actin gene expression and calculated using the 2 -ΔΔCt method.
Bioinformatic analysis To identify enriched gene ontology (GO) terms, we used DAVID web based bioinformatics tool. A GO enrichment test was performed with a cut-off; raw p value >2.00E, false discovery rate (FDR) >2.5E. In addition, the calcium specific functional cluster was conducted with DAVID. Heat-map visualizations were performed with web-based bioinformatics Park et al (2018) Asian-Australas J Anim Sci 31:1507-1515 toll shinyheatmap tool. The amino acid sequences of candidate gene in various species were obtained Ensembl 62. Amino acids were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) (http://www.ebi.ac.uk/ Tools/ msa/muscle/).
Statistical analysis Statistical analysis was performed using Student t-tests in the SAS software (ver. 9.3; SAS Institute, Cary, NC, USA). Significant differences among the different groups were analyzed using the general linear model in SAS. Differences among treatments were deemed to be significant when p<0.05.
RESULTS
Identification of differentially expressed genes during myotube differentiation When QM7 cells were differentiated under the differentiation conditions, QM7 cells were gradually differentiated to myotubes during 3 days (Figure 1A ). In addition, the areas of the differentiated myotubes constantly increased according to days of myotube differentiation (Figure 1B ). To investigate the muscle differentiation-related genes, we conducted highthroughput RNA sequencing analysis for the undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Based on the sequencing data, we aligned and sorted-out 6,687 genes. Among the expressed transcripts, we calculated the expression levels of all genes from each undifferentiated or differentiated sample (day 1 to day 3 of differentiation periods). In Venn diagram of the comparative analysis, we identified 151 DEGs (cutoff; fold change >2, average of normalized read counts (Log2) >2, p value <0.01) including 123 up-and 28 down-regulated genes (Figure 2A ). We also conducted the comparative analysis of DEGs between day 1 to day 3 of differentiation periods and show the Venn diagram in Figure 2B . By comparing with the undifferentiated QM7, 37 and 36 transcripts were identified as up-and down-regulated genes, respectively after day 1 of myotube differentiation. In day 2 of differentiation, 29 and 15 genes were up-and down-regulated. Lastly, 86 and 47 were up-and down-regulated in day 2 of differentiation.
Functional annotation and selection of calcium-related differentially expressed genes Based on mRNA sequencing data, we clustered muscle differentiation-related genes and summarized biological process gene ontology of DEGs in QM7 cells after myotube differentiation (Table 1 ). The most significant enriched terms were 'skeletal muscle contraction' , 'negative regulation of calcium ion transmembrane transporter activity' and 'branch elongation of an epithelium' (Fold enrichment = 15.09, p value = 2.17E-03, FDR = 4.93E-02). In the result of biological process GO of DEGs, the calcium ion-related process was one of the significant enriched terms. To investigate calcium ion functions during muscle differentiation, we sorted calcium-related genes based on biological process GO (Table 2 ). Calciumspecific biological process GO analysis was conducted and 9 calcium-related specific terms were summarized for mus- cle differentiation genes. As a result, we obtained 35 calciumrelated genes in muscle differentiation and compared using Heatmap visualization to examine their expression patterns in each analysis (Figure 2 ). The expression patterns of calciumrelated genes were gradually up-or down-regulated during 3 days of myotube formation (Figure 2 ).
Evolutionary analysis and string analysis of calciumrelated differentially expressed genes To investigate the evolutionary relationships of calcium-related candidate DEGs, we extracted and compared amino acid sequences from eight species in vertebrata (cow, human, duck, mouse, rat, dog, cat, and chicken) regarding two candidate genes, myosin light chain 1 (MYL1) and caveolin 3 (CAV3). When we conducted multiple alignment with MYL1 and CAV3, EFh_PEF super family domain in MYL1 and Caveolin domain in CAV3 showed higher identity (Figure 3 , solid box). Therefore, we suggest that calcium-related domains were highly conserved in the candidate genes and these domains would be interactive with calcium ions during muscle differentiation. In addition, we conducted string analysis among seven calcium related candidate genes (Figure 4 ). As a result, it was confirmed that four genes (MYL1, MYL3, troponin T2, cardiac type [TNNT2], troponin C1, slow skeletal and cardiac type [TNNC1]) interacted with each other.
Validation of calcium-related transcript expression in QM7 cells during myotube differentiation To validate calcium-related transcript expression in muscle differentiation, we selected and analyzed seven genes which were gradually increased according to days of myotube differentiation periods. Similar to RNA sequencing data, the expression patterns of calcium-related transcripts were gradually and highly increased depending on days of myotube differentiation (Figure 5 ). The expression level of MYL3, TNNC1, and phospholamban (PLN) transcripts were higher (<20 folds) than those of undifferentiated QM7 cells while the transcription levels of MYL1, CAV3, calsequestrin 2 (CASQ2) were dramatically increased ranging from 90 to 200 fold during muscle differentiation process (Figure 5 ). These results may indicate that calcium-related genes such as TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2, and TNNT2 play a critical role in muscle differentiation in QM7 cells.
DISCUSSION Cell proliferation, differentiation and cell death are commonly dependent on many signaling pathways controlled by alterations of intracellular calcium . In the previous study, calcium ions were utilized for regulatory molecule and signaling molecule in muscle . It is well-known that calcium
(A) (B) plays a role in muscle contraction-relaxation and mechanism of muscle contraction . In addition, many studies reported the biological functions of calcium ions are not only for mus-cle contraction but also for regulation of energy metabolism through ATP supply . Although the calcium ion plays an important role in muscles, the biological effects of calcium during muscle differentiation have not been well studied. Previous researches indicated that intracellular calcium acted an important role in muscle-specific gene expression, and calcium dependent transcriptional pathway was closely related to skeletal muscle hypertrophic growth . Other studies showed the results of calcium effects on muscle differentiation in frog and rat . In these studies, one of the calcium binding proteins, parvalbumin was identified in myogenesis of frog and rat and detected particularly during the differentiation process of myotomal muscle . Heizmann and Strehler also presented experimental results suggesting the physiological role of parvalbumin in chicken muscle cells. In myotube development of chick embryos, the calcium regulatory system is modulated during myofibiliogenesis and expression level of avian calsequestrin homolog containing calcium storage capacity increased 10-fold before myoblast fusion . These results suggest a biologically significant effect of calcium on muscle differentiation. In this study, we identified calcium-related genes which presented the up-regulated patterns during myotube differentiation and investigated the binding structure with the highly conserved domains between various species (Figure 3 ). The expression of MYL1 gene increased in the myotube differentiation-dependent manner and was significantly detected at an early stage of fast twitch fiber differentiation in zebrafish embryo . The higher similarity of EFh_PEF super family domain sequences in MYL1 and CAV3 gene might be evolutionary evidence of muscle formation in vertebrates. When two or more genes have been derived from a common ancestor, these genes are called orthologous genes and contain a similar domain. These orthologous genes may perform a similar function and so could be evolutionary evidence . Calsequestrin (CASQ) is a main calcium binding protein located within the lumen of sarcoplasmic reticulum . A recent report showed that CASQ regulated a RyR channel are closely related to development of skeletal muscle . Particularly, Schwartz and Kay reported that the expression level of CASQ2 considerably increases immediately before myoblast fusion. The regulatory function of TNNC1 (also known as troponin-C type 1) which is one of calcium binding proteins was reported as calcium ion release from the sarcoplasmic reticulum bind troponin subunits (TnC). After binding of calcium ion with troponin subunits, muscle initiates the contraction. Similarly, this modulatory machinery was also observed during myoblast fusion in quail . Collectively, the results presented indicate that calcium-related candidate genes play important roles in avian muscle differentiation. Figure 1 . 1 Figure 1. Myotube differentiation of QM7 myoblasts. (A) Morphology of QM7 cells during differentiation periods. QM7 cells transformed into myotubes (arrows) during differentiation. (B) Comparison of differentiated areas during differentiation periods. Percentages of differentiation areas were measured by calculating the occupied myotube area in total areas during myotube differentiation.
Figure 2 . 2 Figure 2. The Venn diagrams and heat map for differentially expressed genes (DEGs) during myotube differentiation. (A) Enhanced DEGs during myotube differentiation in QM7 cells (white bars, >2-fold down-regulated genes; black bars, >2-fold up-regulated genes; gray bars, not differentially expressed). (B) The three pie charts displayed the compositions of the DEGs during myotube differentiation periods (day 1 to day 3) compared to the undifferentiated QM7 cells. (C) Heatmap analysis of calcium-related DEGs during myotube differentiation periods (day 1 to day 3) compared to the undifferentiated QM7 cells. Color from green to red indicates low to high expression.
Park et al (2018) Asian-Australas J Anim Sci 31:1507-1515
Figure 4 . 4 Figure 4. String analysis of calcium-related candidate genes processed by RNAsequencing data. Protein-protein interactions were analyzed with the STRING software. In the network, proteins were represented as nodes.
Figure 3 . 3 Figure 3. Comparative alignments of amino acid sequences of MYL1 and CAV3 gene which were highly expressed in myotube differentiation process. EFh_PEF super family domain of MYL1 gene (A) and Caveolin domain of CAV3 gene (B) was highly conserved in various species. The amino acid sequences were aligned by the MUSCLE method in GENEIOUS program. Each conserved domain was marked by solid box. MYL1, myosin light chain 1; CAV3, caveolin 3.
Figure 5 . 5 Figure 5. Expression patterns of calcium-related genes during myotube differentiation. Relative expression levels of candidate genes were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). (n = 3, p<0.005). Quantitative analysis was performed using the 2 -ΔΔCt method by normalization with beta-actin gene.
Table 1 . 1 Biological process gene ontology (GO) analysis of differentially expressed genes (DEGs) which were specifically up-regulated in myotube differentiation QM7 cells (cutoff; raw p value>2.00E, false discovery rate (FDR) >2.5E) GO biological process Expected Fold enrichment Raw p value FDR Skeletal muscle contraction 0.2 15.09 2.17E-03 4.97E-02 Negative regulation of calcium ion transmembrane transporter activity 0.2 15.09 2.17E-03 4.97E-02 Branch elongation of an epithelium 0.2 15.09 2.17E-03 4.97E-02 Gland morphogenesis 0.85 9.39 7.56E-06 5.80E-04 Positive regulation of endothelial cell migration 0.74 8.13 2.12E-04 8.11E-03 Intrinsic apoptotic signaling pathway in response to DNA damage 0.77 7.83 2.53E-04 9.41E-03 Organ growth 0.91 7.7 8.40E-05 3.86E-03 Cellular response to amino acid stimulus 0.65 7.65 9.15E-04 2.62E-02 Lung alveolus development 0.65 7.65 9.15E-04 2.61E-02 Regulation of morphogenesis of a branching structure 0.65 7.65 9.15E-04 2.60E-02 Cellular response to metal ion 1.02 5.87 9.71E-04 2.75E-02 Myeloid cell homeostasis 1.22 4.91 2.20E-03 4.96E-02 Ossification 2.33 4.72 5.06E-05 2.64E-03 Extracellular matrix organization 2.39 4.61 6.17E-05 3.07E-03 Glial cell differentiation 1.65 4.25 2.05E-03 4.74E-02 Regulation of T-cell activation 2.36 4.24 2.46E-04 9.22E-03
Table 2 . 2 Biological process gene ontology (GO) terms of calcium-related differentially expressed genes (DEGs) during myotube differentiation in QM7 cells GO term Gene count Gene symbol Calcium channel complex 10 CASQ2, CAV3, CACNA1S, CACNA2D1, CACNA1B, CACNA1D, ANXA5, PDE4B, CACNG3, CACNB4 Calcium channel activity 8 CAV3, CACNA1S, CACNA1B, CACNA1D, RASA3, TRPV1, SLC24A2, CACNB4 Calcium mediated signaling 15 CASQ2, PLN, ATP2A2, CLIC2, HDAC4, CACNA1D, RCAN3, TOX3, SLC9A1, LRRK2, KDR, ATP1B1, TPCN3, TRPV1, GSTO1 Calcium ion binding 16 MYL1, MYL3, TNNC1, CHIA, NELL2, DAG1, CAPN1, CDH2, VLDLR, CANX, CIB1, MYL9, ANXA2, EFHD1, SPTAN1, FBLN5 Calcium ion import 11 CACNA2D1, ATP2A2, WNT5B, FKBP1A, CACNA1D, RASA3, CHERP, ANXA2, VDAC2, TPCN3, TRPV1 Calcium ion homeostasis 29 CASQ2, PLN, CAV3, GRIA1, BOK, SLC8A3, SMAD3, ATP2A2, STIM1, ATP2B1, F2, HCRTR2, CRY2, FKBP1A, RASA3, CHERP, PTH1R, VAPB, CD24, ATP1B1, GNB1, ANXA7, ATP13A2, GALR2, F2RL1, TPCN3, TRPV1, SLC24A2, CACNB4 Response to calcium 22 CASQ2, TNNT2, CAPN3, SPARC, CPNE2, STIM1, DUSP1, ENTPD6, ANXA11, ANXA5, IL6, SEC31A, ANXA7, KCNIP2, PPP2CA, CCND1, WNT5A, ALOX5AP, PPIF, RANBP1, SLC25A13, INHBB Calcium ion export 2 SLC8A3, ATP2B1 Detection of calcium 3 CASQ2, STIM1, KCNIP2
|
10.5070/t410159759
|
cc-by
| null | null |
openalex
|
Stephanie Fetta's Shaming into Brown is a much-needed contribution to, and beyond the field of Latinx Literatures/Latinx Studies, as Fetta's examination of hegemonic processes of racialization affirms the pivotal role of the body in current understandings of race, ethnicity, and Latinx identities despite the traditional Cartesian mind/body binary. To these ends, Fetta builds on Greek etymology to recognize the soma as "a psychophysical and emotional register of our subjectivity" (xiii), and draws on multiple fields-namely, "Latin@/x studies, literary and critical theories, social psychology, sociology, and critical race studies . . . cognitive science, neuroscience, and social epidemiology" (23)-to propose "somatic analysis, or the semiotic study of somas as a method" (9). Applying her methodology to the study of various Latinx literary texts, Fetta demonstrates the role embodied and somatic experiences play in one's reading and writing practices; experiences of racialization and racism; and ultimately, on current understandings of race, ethnicity, and (the) Latinx/Hispanic Self. It is essential to note the distinction Fetta makes between the soma or somatic expressions compared to our body image and language. While we may learn how to control our body image and language, as common scenarios such as a job interview demonstrate, somatic expressions such as sweaty hands or a pounding heart will truly reveal the responses of our soma (33) (34) (35) . Fundamental to Fetta's study is her use of the term racialization rather than race, as Shaming into Brown critiques current understandings of race as biological and demonstrates that race is but a social construct. Fetta's study focuses instead on what she calls processes of racialization and their consequent racism, defining racialization as "an entrenched U.S. cultural practice of using phenotype and performative markers to designate a subcategory of people as separate and inferior" (3). Further, Fetta insists on using the collective we through her analysis, rather than the individual I typical of scholarly discourse, because "we all participate in racialization in some way" (xix). These processes of racialization and their somatic effect become interpersonal transactions of social power enacting intersectional racial shaming on Latinx individuals and others. This is because, as Fetta condemns, U.S. racial hierarchies are rooted on the normalized shame forced onto the soma through processes of intersectional racialization. With this groundbreaking transdisciplinary scholarly contribution, Fetta urges scholars to question the concept of race as currently understood to instead examine the great impact of social racialization processes onto the soma, as especially illustrated through various literary texts. Fetta weaves her methodology and somatic literary analysis with narrations of her own personal experiences since childhood, along with her own somatic reactions to current events. Fetta's book becomes, thus, partly autobiographical, while she also invites readers to reflect on their own somatic expressions, demonstrating that, indeed, reading is an embodied act. In fact, as I read Fetta's descriptions of her own somatic reactions to the violent processes of racialization she examines or experienced, I could feel my own somatic reactions-the fear and shame overtaking my body as I was confronted with these racialization processes and their harmful somatic impact. Her book is divided into five chapters, along with a preface and conclusion, where her transdisciplinary approach allows her to closely examine the literary representations of somatic expression, focusing each chapter on different aspects of the soma. The first chapter describes Fetta's transdisciplinary approach along with her somatic expressions as she reads Piri Thomas's autobiographical narrative, Down These Mean Streets, and the negotiations of his racial-ethnic identity as a black New York-Puerto Rican. Acknowledging the complexity of her somatic literary analysis, and the fact that most readers may not be knowledgeable of the different fields mentioned above, she continues to explain and expand her methodology throughout the next chapters. In Chapter 2, Fetta focuses on the role vision, audition, and smell play in somatic experiences of racial shaming as portrayed in Yxta Maya Murray's novel Locas (sight), Nelly Rosario's Song of Water Saints (smell), and Angelo Parra's play Song of the Coquí to study hearing and racializing reactions to Spanish accents. Chapter 3 further builds on the methodological approach articulated in Chapters 1 and 2 to analyze The Autobiography of a Brown Buffalo, a novel by Oscar "Zeta" Acosta. In this chapter, Fetta articulates the effects of intersectional racial shaming had on Oscar and his body, specifically, his stomach, somatically materialized with ailments such as ulcers, nausea, vomiting, or constipation (71). This chapter is especially enlightening, as Fetta draws attention to scientific studies highlighting the role of this internal organ in knowledge creation and communication, which we indeed tend to recognize with common sayings such as "I had a gut feeling" (72). Racial shaming is internalized in Oscar's gut, leading him to be chronically ill-a literary representation of the high impact processes of racialization has on the soma and the Self. In Chapter 4, Fetta analyzes Octavio Solís's play Lydia, which has been nominated for a Pulitzer Prize, and the representation of "the ugly soma" (90). Fetta not only focuses on the published script but also on a representation of the play she attended-a literary analysis that also centers her own somatic expressions when encountering Solís's portrayal of disability in connection to gender, homophobia, and undocumented labor. Through her somatic analysis, Fetta highlights the power of theater productions, and especially of Lydia, to center (on) the body. This chapter is also informed by theater scholarship and acting theories and methods, which contribute to Fetta's somatic analysis and the crucial distinction between the soma and the body image represented by the acting body (the actor's) and the acted one. Finally, Chapter 5 pays attention to Andres Montoya's The Ice Worker Signs and Other Poems, a conversion narrative written in verse. Fetta examines Montoya's representation of "the blessed soma, the conversion narrative, and shame" (125), introducing Sophyology into literary analysis, and studying the historical role of Puritanism in racialization processes and racism to the present day. In this way, Fetta's interweaving of literary somatic analysis with cross-disciplinary scholarship and personal accounts demonstrate the social role somatic shaming plays in processes of intersectional racialization-the Shaming into Brown that titles her book-and therefore how social articulations of race, ethnicity take place as Latinx peoples are transformed by, and also transform others, through these racialization processes: a complex demonstration of the pressing need to engage with the soma and its multiple (literary) manifestations to better understand Latinx experiences and current U.S. understandings race relations. Fetta
|
10.4108/eai.13-7-2018.159945
|
cc-by
| null | null |
openalex
|
This paper does the study on the performance of the physical layer secrecy of nonorthogonal multiple access (NOMA) in downlink cooperative. The given system includes one source, multiple legitimate user pairs in the form of an eavesdropper. By applying the decode-and-forward (DF) scheme, a good user will take the information from the source to send it to the bad user in every pair, we assume that the eavesdropper will spend effort to decode the message from the bad user. To enhance the secrecy performance of given system, the artificial noise cooperative transmission scheme named ANCOTRAS is suggested. To assess the performance of the suggested scheme, we obtained the lower bound and exact closed-form expressions of secrecy outage probability by implementing statistical characteristics of signal-to-noise ratio (SNR) and signalto-interferenceplus-noise ratio (SINR). Furthermore, the secrecy performance of given system is studied basing on key parameters (including the power allocation ratio), average transmit power and amount of user pair for verifying the suggested scheme. At the end, the accuracy of final analytical outcome is reassured by using the Monte-Carlo simulation results.Introduction In the recent years, the portable devices with wireless features and integrated smart operation system such as smartphones, smart watches or smart home devices became an integral part of human society, all thanks to their flexibility and multi-function. It also leads us to the compromising booming of fifth-generation network (5G) as a replacement for 4G. The reason for this changes are because the growing number of wireless devices also go hand in hand with the need for low cost and low latency networks, which the 4G and earlier generation networks, using tradition orthogonal multiple access (OMA) such as FDMA, TDMA, and CDMA, cannot meet because of the limited channel resource and the spectral efficiency loss. To adapt to the requirements of 5G network, the non-orthogonal multiple access (NOMA) technique is the most promising one requirements . With the ability in serving multiple users in the same period of time, frequency or code, NOMA can make sure that the spectral efficiency can be maintained. Besides, the equality of user can be increased in NOMA in comparison with OMA. Furthermore, the cooperative relaying technique which can be applied to NOMA networks can enhance the effectiveness and widen the covered area of the wireless networks [6, 7] and it also can attract the attention of other researchers on this topic . In specific, a cooperative NOMA transmission scheme is suggested in the work to V.L. Nguyen et al. explore the previous information in NOMA system, in which the better channel condition helps the users decode the message from others. For user having poor connection, the given information takes the form of the relays to increase the reception reliability. The NOMA cooperative relaying system is studied in the work which also suggested the most suitable relay selection (BRS) scheme. In the outcome, it could be seen that in the case of increasing number of relays, the NOMA-based BRS acquired more rate gain than the traditional BRS. The [10, 11] study is NOMA cooperative relaying system with energy harvesting and provided another communication protocol for cooperative NOMA system. In them, the writer give the conclusion that in the weak transmission conditions their protocols can deal with the lack of direct link between the paired users. Moving to the eavesdropping issue, while the information security is becoming more and more vital in the information age because the value of our confidential information in using for illegal activities, our information become more vulnerable than ever before because of the broadcast nature of wireless communications. The activities of eavesdropping by wiretappers are hard to be discovered and convicted while information transmission between transceivers can be eavesdropped with not many efforts. Although there are some provided solutions such as RSA and DES but most of them are used at the higher layers such as application or network layers. In addition to it, they are also limited in error-free condition of the link between the transmitter and receiver and the ability of eavesdroppers are assumed in the low computing power level and lacking of efficient algorithms . In the given situation, the physical layer secrecy (PLS) is suggested to accomplish the secure transmission to improve the security level of wireless networks by using the dynamic characteristics of the wireless channels . The secrecy ability of NOMA network can be increased by applying this approach. But the involvement of successive interference cancellation (SIC) in NOMA technique can differentiate it from conventional OMA technique. In the recent time, the physical layer secrecy (PLS) of NOMA relaying networks attracted a lot of works. For example, the investigated the PLS of a downlink NOMA system with the assuming that the required users quality of service (QoS) to deploy NOMA and all channels undergo Nakagami-m fading. From the outcome, it's can be seen that, the secrecy performance of the overall communication process can be improved when the difference in the level of priority between legitimate users is low. The analyzed the secrecy performance of a two-user downlink NOMA system. The researcher included two single-input single-output and multiple-input single-output systems which is different in transmit antenna selection (TAS). The secrecy outage probability (SOP) for different TAS schemes is also expressed in precise and estimated closed form. The outcome is that the SOP for the far user with fixed power allocation scheme drop when the transmit power increase, as the transmit power beyond the threshold, and, after that, it touch the floor with the rising of the interference from the near user. In , the authors studied the PLS of large-scale NOMA networks through the SOP. In this article, the locations of NOMA users and eavesdroppers were mapped by using stochastic geometry approaches. And the authors in produced the artificial noise (AN) at the base station to increase the security of a beamformingaided multiple-antenna system. They suggested The secrecy beamforming scheme for multiple-input singleoutput non-orthogonal multiple access (MISO-NOMA) system, in which, confidential information of two NOMA assisted legal users was protected by using AN, so the system secrecy performance is enhanced. In , the PLS for cooperative NOMA systems including amplify-and-forward (AF) and decode-and-forward (DF) protocols is considered. The conclusion showed that AF and DF schemes reach approximately similar secrecy performance and this secrecy performance is separated from the channel conditions between the relay and the poor user. Different from the works above, our work focusses on the PLS of downlink NOMA cooperative relay communication system in combination with AN to increase the PLS performance. To be specific, our major subject is the PLS performance of the NOMA system, in that system, the base station (BS) simultaneously delivers information to user pairs based on powerdomain, then, the information will be forwarded from the better user (user with better channel condition) to the worse user (users with poor channel conditions), assuming that only the worse users are eavesdropped. AN is applied in the BS to blur the eavesdropper to improve the performance of PLS. Our article contributes the ideas below: 1. Suggesting AN with cooperative transmission scheme (ANCOTRAS). 2. Deriving the lower bound and exact closed-form expressions of secrecy outage probability for each user and overall system. 3. Evaluating the PLS performance using tools of secrecy outage probability expressions. 4. Studying the above system behavior in distinct key parameters, including power allocation ratio, average transmit power and the number of user pairs. Enhancing Physical Layer Security for Cooperative Non-Orthogonal Multiple Access Networks with Artificial Noise 5. Comparing between the PLS performance of ANCOTRAS scheme and non-AN scheme to identify the advantages of integrated AN NOMA cooperative system. The rest of this paper is organized as follows. The system model is presented in Section II. Secrecy performance of the considered system is analyzed in Section III. The numerical results are shown in Section IV. Finally, Section V draws the conclusion of our paper.
Network and Channel Models The Fig. 1 illustrated a A cooperative communication system for downlink NOMA. In which, the information is intended to be transmitted to M mobile user's represented as D i (i = 1 <= m < n <= M), by the source S i.e., base station, with the under the presence of an eavesdropper E. In this system, it is ok to split the M users to multiple pairs, such as {D m , D n } , m < n, to perform NOMA , and in the information signal will be exchanged (by forwarding information) between two paired users. This means m th user and the n th user are paired to deploy cooperative NOMA. Using the applying successive interference cancellation (SIC) to cancel the interference and detect the D m 's signal the better user, then, forward the information of the worse user D n . Without loss of generality, assuming that all the channel gains between S and users follow the order of |h SD 1 | 2 <= |h SD 2 | 2 <= ... <= |h SD m | 2 <= |h SD n | 2 <= ... <= |h SD M | 2 are
Phase 1 In this phase, the source S broadcasts information to the M users. The received signals at D m and at D n are y SD m = P S d θ SD m ( √ a m s m + √ a n s n )h SD m + n SD m , ( 1 ) y SD n = P S d θ SD n ( √ a m s m + √ a n s n )h SD n + n SD n , (2) respectively, where n SD m and n SD n are the AWGN with zero mean and variance σ 2 . Let X 1 |h SD m | 2 , Y 1 |h SD n | 2 , X 2 |h mn | 2 , Z 1 |h SE | 2 , and Z 2 |h DmE | 2 . The instantaneous SINR at the n th user to detect s n transmitted from S can be given by γ SD n = a n γ|h SD n | 2 a m γ|h SD n | 2 + d θ SD n = b 2 Y 1 b 1 Y 1 + 1 , ( 3 ) where γ = P S σ 2 is denoted as the average transmit SNR of S -D n link, b 1 = a m γ d θ SD n , b 2 = a n γ d θ SD n . Similarly, the instantaneous SINR at the m th user to detect s n transmitted from S can be written as γ s n SD m = a m γ|h SD m | 2 a n γ|h SD m | 2 + d θ SD m = b 4 X 1 b 3 X 1 + 1 , ( 4 ) where b 3 = a m γ d θ SD m , b 4 = a n γ d θ SD m . At the same time, the received signal at E is given as follows y SE = P S d θ SE ( √ a m s m + √ a n s n )h SE + n SE , ( 5 ) where n SE is the AWGN with zero mean and variance σ 2 E . Due to assuming that the eavesdropper only tries to detect s n , therefore the instantaneous SINR at E is given by γ SE = a n γE |h SE | 2 a m γE |h SE | 2 + d θ SE = b 6 Z 1 b 5 Z 1 + 1 , ( 6 ) where b 5 = a m γE d θ SE , b 6 = a n γE d θ SE .
Phase 2 In phase 2, D m uses the power P Dm to forward s n to D n and S and, at the same time, uses the power P S to broadcast an AN to users and eavesdropper. The instantaneous SINR at D n in the second phase is as follows: γ mn = U 1 = c 1 X 2 c 3 Y 1 + 1 , (7) where c 1 = γD m d θ mn , c 3 = γ d θ SDn , γD m = P D m σ 2 . Equally, the instantaneous SINR at E in this phase is given by γ DmE = U 2 = c 2 Z 2 c 4 Z 1 + 1 , ( 8 ) where c 2 = γD m E d θ DmE , c 4 = γE d θ SE , γD m E = P D m σ 2 E . To identify the advantages of ANCOTRAS protocol, a study on the case of non-AN also is done. In this, the instantaneous SNR at D n in the phase 2 is as follows: γ mn = P D m |h mn | 2 σ 2 d θ mn = c 1 X 2 , (9) Similarly, the instantaneous SNR at E in the last phase is given by γ DmE = P Dm |h DmE | 2 σ 2 d θ DmE = c 2 Z 2 , ( 10 ) Considering i.i.d. Rayleigh channels, the channel gains |h SDm | 2 , |h SDn | 2 , |h SE | 2 and |h mn | 2 follow exponential distributions with parameters λ SDm , λ SDn , λ SE and λ mn , respectively. In order statistics, the probability density function (PDF) and the cumulative distribution function (CDF) of U , where U ∈ {X 1 , Y 1 }, are respectively given by f U (x) = M! (M -i)!(i -1)! 1 λ SDi i-1 k=0 C i-1 k (-1) k e -x(M-i+k+1) λ SDi (11) F U (x) = M! (M -i)!(i -1)! i-1 k=0 C i-1 k (-1) k × 1 M -i + k + 1 1 -e -x(M-i+k+1) λ SDi . ( 12 ) where i ∈ {m, n} The PDF and CDF of V , where V ∈ (X 2 , Z 1 , Z 2 ) are respectively expressed as f V (x) = 1 λ e -x λ ( 13 ) F V (x) = 1 -e -x λ ( 14 ) where λ ∈ {λ mn , λ SE , λ DmE }. Adding more calculation, we derive CDFs and PDFs of γ s n SD m , γ SD n , γ SE , γ mn , γ D m E . Drawn from above outcomes, The CDF of γ SDn are calculated as below: F γ s n SDm (x) = P r b 4 X 1 b 3 X 1 + 1 < x (a) = F X 1 x b 4 -b 3 x = M! (M -m)!(m -1)! m-1 k=0 C m-1 k (-1) k × 1 M -m + k + 1 1 -e -(M-m+k+1)x λ SDm , ( 15 ) F γ SDn (x) = P r b 2 Y 1 b 1 Y 1 + 1 < x (b) = F Y 1 x b 2 -b 1 x = M! (M -n)!(n -1)! n-1 k=0 C n-1 k (-1) k × 1 M -n + k + 1 1 -e -(M-n+k+1)x λ SDn . ( 16 ) Given that step , respectively. Equally, the CDF and PDF of γ SE are derived respectively as belows: The CDF of the γ mn is calculated as follows F γ SE (x) = P r b 6 Z 1 b 5 Z 1 + 1 < x (c) = F Z 1 x b 6 -b 5 x = 1 -e - x λ SE (b 6 -b 5 x) , ( 17 ) f γ SE (x) = b 6 λ SE (b 6 -b 5 x) 2 e F γ mn (x) = Pr c 1 X 2 c 3 Y 1 + 1 < x = ∞ 0 F X 2 x(c 3 y + 1) c 1 f Y 1 (y)dy = 1 - M! (M -n)!(n -1)! 1 λ SDn n-1 k=0 C n-1 k (-1) k × ∞ 0 e - x(c 3 y+1) c 1 λ mn - y(M-n+k+1) λ SDn dy = 1 - M! (M -n)!(n -1)! oversetn -1 k=0 C n-1 k (-1) k × c 1 λ mn c 3 λ SDn x + c 1 λ mn (M -n + k + 1) e -x c 1 λ mn . ( 19 ) Similarly the CDF of the γ D m E is given by F γ D m E (x) = Pr c 2 Z 2 c 4 Z 1 + 1 < x = ∞ 0 F Z 2 x(c 4 y + 1) c 2 f Z 1 (y)dy = 1 - 1 λ SE ∞ 0 e - x(c 4 y+1) c 2 λ DmE - y λ SE dy = 1 - c 2 λ DmE c 4 λ SE x + c 2 λ DmE e - x c 2 λ DmE . ( 20 ) The PDF of the γ D m E is expreesed as follows: f γ D m E (x) = c 2 c 4 λ DmE λ SE (c 4 λ SE x + c 2 λ DmE ) 2 + 1 (c 4 λ SE x + c 2 λ DmE ) × e - x c 2 λ DmE . ( 21 )
Analyzing Secrecy Performance This part analyzed secrecy performance is analyzed in term of secrecy outage probability (SOP). SOP is an crucial performance metric which is applied to describe the secrecy performance of a wireless communication system. In this paper, the secrecy performance is investigated in terms of SOP at S and at D m with the assumption that E wants to take out the message of D n .
Preliminaries The instantaneous capacities of the legitimate channel and eavesdropper channel can be respectively defined by C M = B log(1 + γ M ), ( 22 ) C N = B log(1 + γ N ), ( 23 ) where B is bandwith (Hertz), γ M ∈ {γ SD m , γ SD n , γ mn }, γ N ∈ {γ SE , γ D m E }. The instantaneous secrecy capacity for S -D m , S -D n and D m D n are given by C S 1 = C γ s n SD m -C γ SE + = α log 2 1 + γ s n SD m 1 + γ SE , γ s n SD m > γ SE 0, γ s n SD m <= γ SE , ( 24 ) C S 2 = C γ SD n -C γ SE + = α log 2 1 + γ SD n 1 + γ SE , γ SD n > γ SE 0, γ SD n <= γ SE , ( 25 ) C S 3 = C γ mn -C γ D m E + = (1 -α) log 2 1 + γ mn 1 + γ D m E , γ mn > γ D m E 0, γ mn <= γ D m E , ( 26 ) C S 4 = C γ mn -C γ D m E + = (1 -α) log 2 1 + γ mn 1 + γ D m E , γ mn > γ D m E 0, γ mn <= γ D m E (27) respectively. Here, for simplicity we assume B = 1Hz. SOP is defined as the probability that the instantaneous secrecy capacity falls below a predetermined secrecy rate threshold R S > 0, given by SOP = P r(C S < R S ). Notice that, in this considered system we only consider the case of the eavesdropper tries to hear the message of D n at S and at D m . In the next subsection, we present the calculation of the SOPs at S and at D m .
Secrecy outgage probability at S The SOP at S of S -D n link (SOP 1 ) can be calculated as follows: SOP 1 = Pr(C S 1 < R S ) = P r 1 + γ SD n 1 + γ SE < 2 R S /α = 1 -P r γ SE < 1 + γ SD n -2 R S /α 2 R S /α . ( 28 ) Because the equation ( 28 ) is intractable to obtain the closed-form expression, only the lower bound of SOP 1 is obtained here. From the equation (3), γ SD n < a n a m can be seen, so the lower bound of SOP 1 should be as follows: (30) SOP 1 > SOP 1 lower = 1 -CDF γ SE 1 + a n a m 2 R S /α -1 = e
Secrecy outgage probability at D m In this situation, the secrecy outage event happens when s n canâĂŹt be noticed by D m or D m can detect s n but the secrecy capacity is under the secrecy threshold. As a result, the SOP at the D m can be calculated as below: -SOP at D m with AN. SOP 3 = Pr(γ s n SD m < γ t ) + Pr(γ s n SD m > γ t , C S 3 < R S ) = Pr b 4 X 1 b 3 X 1 + 1 < γ t + 1 -Pr b 4 X 1 b 3 X 1 + 1 < γ t × Pr 1 + γ mn 1 + γ D m E < 2 R S 1-α . ( 31 ) Proposition 1. Under Rayleigh fading, the SOP of the link D m -D n with AN is given by SOP 3 = Φ 1 + (1 -Φ 1 )(1 - M! (M -n)!(n -1)! × n-1 k=0 C n-1 k (-1) k (Φ 2 + Φ 3 )), (32) where Φ 1 = M! (M-m)!(m-1)! m-1 k=0 (-1) k C m-1 k 1 M-m+k+1 × 1 -e - γ t (M-m+k+1) (b 4 -b 3 γ t )λ SDm , γ t < a n a m , 1, γ t > a n a m , Φ 2 = c 1 c 2 c 4 λ DmE λ SE λ mn e -2 R S (1-α) -1 c 1 λ mn × ce dμ c Γ (0, dμ c ) (ad -bc) 2 - ce bμ a Γ (0, bμ a ) (ad -bc) 2 + μe bμ a Γ (-1, bμ a ) a(ad -bc) 2 , Φ 3 = c 1 λ mn e -2 R s -1 c 1 λ mn 1 ad -bc e b a μ Γ (0, b a μ) - 1 ad -bc e d c μ Γ (0, d c μ) . Denoted that, a = c 4 λ SE , b = c 2 λ DmE , c = c 3 λ SDn 2 R S /(1-α) , d = c 3 λ SDn (2 R S /(1-α) -1) + c 1 λ mn (M - n + k + 1), μ = 2 R S /(1-α) c 1 λ mn + 1 c 2 λ DmE . Proof. See Appendix A. -SOP at D m without AN. SOP 4 = Pr(γ s n SD m < γ t ) + Pr(γ s n SD m > γ t , C S 4 < R S ) = Pr b 4 X 1 b 3 X 1 + 1 < γ t + 1 -Pr b 4 X 1 b 3 X 1 + 1 < γ t × Pr 1 + c 1 X 2 1 + c 2 Z 2 < 2 R S 1-α . ( 33 ) Proposition 2. Under Rayleigh fading, the SOP of the link D m -D n without AN is given by SOP 4 = Φ 1 + (1 -Φ 1 )Φ 4 , ( 34 ) where Φ 4 = 1 - c 1 λ mn 2 R S /(1-α) c 2 λ DmE + c 1 λ mn e -2 R S /(1-α) -1 c 1 λ mn . Proof. See Appendix B.
Secrecy outgage probability of overall system As a result of D n applying selection combining scheme, the instantaneous secrecy can be calculated as belows: C S 5 = C S 1 , γ s n SD m < γ t , max{C S 1 , min{C S 2 , C S 3 }}, γ s n SD m > γ t , ( 35 ) The SOP of overall system is given by 36 ) Proposition 3. Under Rayleigh fading, the SOP of overall system with AN is given by SOP 5 = Pr(C S 5 < R S ) = Pr(γ s n SD m < γ t )SOP 1 + Pr(γ s n SD m > γ t , max{C S 1 , min{C S 2 , C S 3 }} < R S ). ( SOP 5 = F γ s n SDm (γ t ).SOP 1 + SOP 1 .SOP 2 .SOP 3 × 1 -F γ s n SDm (γ t ) > F γ s n SDm (γ t ).SOP 1lower + SOP 1lower .SOP 2lower .SOP 3lower × 1 -F γ s n SDm (γ t ) (37) Proof. See Appendix C. Similarly, SOP of the overall system without AN is given by SOP 6 = F γ s n SDm (γ t ).SOP 1 + SOP 1 .SOP 2 .SOP 4 × 1 -F γ s n SDm (γ t ) > F γ s n SDm (γ t ).SOP 1lower + SOP 1lower .SOP 2lower .SOP 4lower × 1 -F γ s n SDm (γ t ) (38)
Nummerical results and disscussion In this section, the PLS performance of ANCOTRAS protocol for this considered cooperative NOMA system is analyzed by numerical results. And our analytical results is also verified using Monte-Carlo simulation results. The outcomes of SOP of the system at S is illustrated in Figs. 2 and Figs. 3. figures displayed that the average transmits SNR from S to user D n ( γ) then the SOP at the S reduces when the distance from S to the eavesdropper E(d SE ) is increased. That means there are increase in the PLS capability of this model. Besides, in these two results, when we increase the average transmit SNR from S to the E ( γE ), the SOP at S of the system also increases.
Secrecy outage probability at D m Figs. 4 and Figs. 5 depict the results of the SOP of the system at D m . In these two results, the secrecy performance is analyzed in consistent with the changes of the parameters belows: γ, γDm and γE . In these figures, We can see that the SOP at D m reduces when γDm (the average transmit SNR from D m to D n ) increase. From Figs. 4 , we can see that the secrecy performance can be improved if we use AN from S to D n ( γ at second phase). Similarily, in Figs. 5 it is clear that the average transmit SNR of AN is increased from S to E ( γE ), the secrecy performance is improved. However, introducing AN when the average transmit SNR is low ( γE = 10 dB), the security performance of the system will not be improved compared to the case of not using AN. To be more specific, in this case, we should use γE The survey results in Figs. 6 showed that the SOP at D m of the system reduce along with the increase in γDm and reduce in γDmE . This result settles once again that using AN the will increases the PLS performance of the system. Seeing the changes in the figures of users (M), which using AN, in figs. 7, it is showed that the SOP at the D m of system decreases when M increases. Using he formula of SOP 3 , the conclusion is that, C S 3 increases when M increase so SOP 3 decreases. Particularly, the simulation results in figs. 7 showed that with 3 sequential M values: 4,6 and 8, SOP 4 do not experience major change. So it can be seen that when the system according to the changes of M and γDm is
Secrecy outage probability overall system Fig. 8 illustrates the SOP of the overall system. The result displays that the higher the average transmit SNR from D m to D n ( γDm ) is the lower SOP of the overall system is observed. It means that secrecy performance will rise. On another side, it can be seen that when the average transmit SNR from S to D n ( γ) rise, then SOP of the overall system with reduce, but, if γ is big enough (particularly when γ >= 20 (dB) in this figure) then SOP of the overall system with AN will rise. In phase 2, γ is the average transmit SNR of the AN, so if γ is greater than a appropriate value (particularly when γ >= 10 (dB) in this figure) the security performance of the system with AN will reduce. We can see the SOP of the overall system with AN reduced when increasing the average transmit SNR from S to E ( γE ) in Fig. 9 . In this figure, the increase of γE go hand in hand with the decrease of SOP. However, γE have to be at suitable (particularly when γE >= 10 (dB) in this figure), the security performance of the system with AN better than that the security performance of the system without AN.
Conclusion In this work, we inspected the secrecy performance of a downlink cooperative NOMA network with artificial noise. Particularly, in this paper, the method of broadcasting artificial noise from the base station is used to interfere the eavesdroppers and we also improved the information security. Besides, new analytical expressions were carried out in terms of the secrecy outage probability to regulate the system secrecy performance. In the meantime,we used the numerical results to confirm the examines. Basing on the analytical and simulation results, it can be settled that the security level of the network model which is suggested in this paper relies on the distance: from 1) base station to the better user; worse users; eavesdropping device and from 2) better users to worse users, eavesdropping devices. Using artificial noise can improve the security performance of the system. As long as appropriate values are used, the securecy performance of the system with AN is better than that without AN. APPENDIX A: Here, we derive the expression of SOP at D m in the case of with AN. SOP 3 = Pr b 4 X 1 b 3 X 1 + 1 < γ t + 1 -Pr b 4 X 1 b 3 X 1 + 1 < γ t × Pr 1 + γ mn 1 + γ D m E < 2 R S /(1-α) = Φ 1 + (1 -Φ 1 ) × ∞ 0 F U 1 (2 R S /(1-α) (1 + y) -1)f U 2 (y)dy = Φ 1 + (1 -Φ 1 ) 1 - M! (M -n)!(n -1)! n-1 k=0 C n-1 k (-1) k × ∞ 0 (c 1 λ mn ) e - (2 R S /(1-α) (1+y)-1) c 1 λ mn / (c 3 λ SDn (2 R S /(1-α) (1 + y) -1) + c 1 λ mn (M -n + k + 1)) × c 2 c 4 λ DmE λ SE (c 4 λ SE y + c 2 λ DmE ) 2 + 1 (c 4 λ SE y + c 2 λ DmE ) × e - y c 2 λ DmE dy = Φ 1 + (1 -Φ 1 )(1 - M! (M -n)!(n -1)! × n-1 k=0 C n-1 k (-1) k (Φ 2 + Φ 3 )), (39) where γ t is the threshold to detect s n and Φ 1 , Φ 2 , Φ 3 are calculated as follows Φ 1 = F X 1 γ t b 4 -b 3 γ t = M! (M-m)!(m-1)! m-1 k=0 (-1) k C m-1 k 1 × 1 -e - γ t (M-m+k+1) (b -b γ t )λ SDm , γ t a a m 1, γ t > a n a m Φ 2 = c λ mn ∞ 0 c c 4 λ λ SE (c 4 λ SE y + c 2 DmE ) 2 e - y c 2 λ DmE × dy c 3 λ SDn (2 R S /(1-α) (1 + y) -1) + 1 λ mn (M -n + k + 1) = c 1 λ mn c 2 c 4 λ DmE λ SE e -2 R S /(1-α) -1 c 1 λ mn × ∞ 0 1 (ay + b) 2 1 cy + d e - 1 c 2 λ DmE + 2 R S /(1-α) c 1 λ mn y dy = c 1 λ mn c 2 c 4 λ DmE λ SE e -2 R s -1 c 1 λ mn ∞ 0 E 1 cy + d e -μy dy + ∞ 0 E 2 ay + b e -μy dy + ∞ 0 E 3 (ay + b) 2 e -μy dy = c 1 λ mn c 2 c 4 λ DmE λ SE e -2 R s -1 c 1 λ mn Figure 1 . 1 Figure 1. System model for secure cooperative NOMA
denoted as the ordered channel gains of the m th user and the n th user. Denote that |h mn | 2 is the channel gains of the links between the m th user and the n th user; |h SE | 2 and |h DmE | 2 are channel gains of the links of S -E and D m -E, respectively. We assume that all the nodes are single-antenna devices and operate in a halfduplex mode. All wireless links are assumed to undergo independent frequency non-selective Rayleigh block fading and additive white Gaussian noise (AWGN) with zero mean and the same variance σ 2 . We denote d SD m , d SD n , d mn , d SE , d DmE as the Euclidean distances of S -D m , S -D n , D m -D n , S -E, D m -E, respectively and θ denote the path-loss exponent.
1 , x < b 4 b 3 13 (a) and (b) are gained by assuming the following condition holds x < b 2 b
4 V 5 . 45 x λ SE (b 6 -b 5 x) . (18) .L. Nguyen et al. EAI Endorsed Transactions on Industrial Networks and Intelligent Systems 06 2019 -08 2019 | Volume 6 | Issue 20| e4 Note that step (c) is obtained by assuming the following condition holds x < b 6 b
5 Enhancing 5 β λ SE (b 6 -b 5 β) , (29) Physical Layer Security for Cooperative Non-Orthogonal Multiple Access Networks with Artificial Noise EAI Endorsed Transactions on Industrial Networks and Intelligent Systems 06 2019 -08 2019 | Volume 6 | Issue 20| e4 where β = 1+ a n a m 2 R S /α -1. Similarly, for SOP at S of S -D m link (SOP 2 = Pr(C S 2 < R S )), the lower bound SOP 2 lower is the same as SOP 1 lower : SOP 2 > SOP 2 lower = e -β λ SE (b 6 -b 5 β) .
4. 1 . 3 Sim: d SE = 3 Figure 2 . 1332 Figure 2. The SOP at the Base Station with a distance change from S to E; d SDm = 1, d SDn = 2, α = 0.3
Figure 3 .Figure 4 . 34 Figure 3. The SOP at the Base Station with a distance change of γ; d SDm = 1, d SDn = 2, d SE = 1, α = 0.3, R s = 0.5
7Figure 5 .Figure 6 . 56 Figure 5. The SOP at the D m with the change of γE ; d SDm = 1, d SDn = 2, d mn = 1, d DmE = 1, α = 0.3
8 Figure 7 .Figure 8 . 878 Figure 7. The SOP at the D m with the change of M; d SDm = 1, d SDn = 2, d mn = 1, d DmE = 1, α = 0.3
c 1 λ mn c 3 λ- 1 c 2 312 SDn (2 R s (1 + y) -1) + c 1 λ mn e λ SE y + c 2 λ DmE ) e 4 λ SE , b = c 2 λ DmE , c = c 3 λ SDn 2 R S /(1-α) , d = c 3 λ SDn (2 R S /(1-α) -1) + c 1 λ mn (Mn + k + 1), μ = 2 R S /(1-α) c 1 λ mn + λ DmE , E 1 = c 2 (ad-bc) 2 , E 2 = -ac (ad-bc) 2 , E 3 = a (ad-bc) , F 1 = -c ad-bc , F 2 = a ad-bc . Substituting Φ 1 , Φ 2 , Φ 3 into (39), we obtain the closed-form expression of SOP for the link D m -D n in the case of using AN. This concludes the proof.
SOP 4 = 9 Enhancing 1 c 1 Substituting Φ 1 , 1 + 2 .I 1 I 2 = 491111212 Pr(γs n SD m < γ t ) + Pr(γ s n SD m > γ t , C S 4 < R S ) = Pr b 4 X 1 b 3 X 1 + 1 < γ t + 1 -Pr b 4 X 1 b 3 X 1 + 1 < γ t × Pr 1 + c 1 X 2 1 + c 2 Z 2 < 2 R S 1-α = Φ 1 + (1 -Φ 1 )Φ4, (40) Physical Layer Security for Cooperative Non-Orthogonal Multiple Access Networks with Artificial Noise EAI Endorsed Transactions on Industrial Networks and Intelligent Systems 06 2019 -08 2019 | Volume 6 | Issue 20| e4whereΦ 4 = Pr X 2 < 2 R S /(1-α) (1 + c 2 Z 2 ) 2 R S /(1-α) c 2 λ DmE + c 1 λ mn e -2 R S /(1-α) -λ mn. Φ 4 into (40), we obtain the closedform expression of SOP for the link D m -D n in the case of without using AN. This concludes the proof.APPENDIX C:SOP 5 = Pr(γ s n SD m < γ t )SOP 1 + Pr(γ s n SD m > γ t , max{C S 1 , min{C S 2 , C S 3 }} < R S ) = F γ s n SD m (γ t )SOP 1 I Pr(γ s n SD m > γ t , C S 1 < R S , min{C S 2 , C S 3 } < R S ) I > F γ s n SD m (γ t ).SOP 1lower = M! (Mn)!(n -1)! SOP 1 . Pr(γ s n SD m > γ t , min{C S 2 , C S 3 } < R S ) = SOP 1 . 1 -Pr(γ s n SD m > γ t , min{C S 2 , C S 3 } > R S ) = SOP 1 . 1 -Pr(γ s n SD m > γ t , C S 2 > R S , C S 3 > R S ) = SOP 1 . 1 -Pr(γ n SD m > γ t , C S 2 > R S ).SOP 3 = SOP 1 .{1 -1 -s n SD m > γ t , C S 2 < R S ) .SOP 3 }(42)
V.L. Nguyen et al.EAI Endorsed Transactions on Industrial Networks and Intelligent Systems
V.L. Nguyen et al.EAI Endorsed Transactions on Industrial Networks and Intelligent Systems
V.L. Nguyen et al.EAI Endorsed Transactions on Industrial Networks and Intelligent Systems
|
10.1186/s40104-018-0230-8
|
cc-by
| null | null |
openalex
|
Background: Extensive degradation of amino acids in the rumen via microbial deamination decreases the postruminal availability of dietary indispensable amino acids. Together with the normal decrease in voluntary dry matter intake (DMI) around parturition in dairy cows, microbial metabolism contributes to a markedly negative balance of indispensable amino acids, including methionine which may be the first-limiting for milk production. The main objective of the current study was to profile changes in major bacterial species with key functions in cellulose and hemicellulose digestion, xylan breakdown, proteolytic action, propionic acid production, lactate utilization and ruminal biohydrogenation in cows supplemented with rumen-protected Methionine (SM; Smartamine M, Adisseo NA, Alpharetta, GA, USA) from -23 through 30 d relative to parturition. Because ~90% of the methionine in SM bypasses the rumen, ~10% of the methionine is released into the rumen and can be utilized by microbes. Results: As expected, there was an increase in overall DMI after parturition (Day, P < 0.05) during which cows consumed on average 19.6 kg/d versus 13.9 kg/d in the prepartum period. The postpartum diet contained greater concentrations of lipid and highly-fermentable carbohydrate from corn grain, which likely explains the increases in the relative abundance of Anaerovibrio lipolytica, Megasphaera elsdenii, Prevotella bryantii, Selenomonas ruminantium, Streptococcus bovis, and Succinimonas amylolytica. Despite similar DMI prepartum, cows fed SM had greater (Treatment × Day, P < 0.05) abundance prepartum of Fibrobacter succinogenes, Succinimonas amylolytica, and Succinivibrio dextrinosolvens. However, the greater DMI in cows fed SM after parturition (19.6 kg/d versus 13.9 kg/d) was associated with lower abundance of Fibrobacter succinogenes (2.13 × 10 -3 versus 2.25 × 10 -4 ) and Selenomonas ruminantium (2.98 × 10 -1 versus 4.10 × 10 -1 ). A lower abundance (Day, P < 0.05) was detected on d 20 compared with d -10 for Fibrobacter succinogenes and Succinivibrio dextrinosolvens. The relative abundance of Butyrivibrio proteoclasticus and Eubacterium ruminantium was stable across treatment and time.Background Extensive degradation of amino acids in the rumen via microbial deamination [2, 3] substantially lowers the postruminal availability of dietary indispensable amino acids (IAA). Together with the normal decrease in voluntary dry matter intake (DMI) around parturition in dairy cows, microbial metabolism contributes to a markedly negative balance of IAA, including methionine which often is the first-limiting amino acid . Therefore, the importance of enhancing the delivery methionine to the small intestine through "rumen by-pass" technologies has been underscored since the early 1960's . The physiologic impact of supplementing dairy diets with rumen-protected methionine (RPM) at lactation stages where needs are the greatest has received close attention worldwide, i.e. improving the overall health, metabolism, and production performance in dairy cows . With the advances in methionine protection technology, a large proportion of protected methionine escapes ruminal degradation but a small fraction of methionine is still released into the rumen and may change the community composition of the rumen microbiota and their metabolism. During the peripartal period, dairy cow diets move from higher-forage content before calving to higher-concentrate diets postpartum to provide the rumen microbial communities with more readily-available energy. As a result, microbial composition changes in favor of generating more volatile fatty acids (VFA) and microbial protein to serve as a fuel and amino acid sources, respectively, for body tissues and milk synthesis . Therefore, nutritional management plays a key role in shaping the microbial ecosystem in the rumen [13, 14] . Despite the continued focus on nutritional management of peripartal cows, little is known about the response of rumen microbes to methionine supplementation. For example, Salsbury et al. and Gil et al. were among the first to observe that unprotected supplemental methionine enhanced ruminal bacteria growth rate and protein synthesis in vitro. Although there is growing evidence that RPM enhances overall dairy cow health and productivity, whether ruminal bacteria composition changes in response to RPM supplementation remains unknown. Therefore, the objective of the current study was to profile changes in major bacterial species with key functions in cellulose and hemicellulose digestion, xylan breakdown, proteolytic action, propionic acid production, lactate utilization and ruminal biohydrogenation in cows supplemented with RPM (Smartamine M, Adisseo NA, Alpharetta, GA) from -23 through 30 d around parturition. Smartamine M is designed to release over 90% of the methionine along the small intestine. Thus, the remaining 10% of the methionine is released into the rumen and can be utilized by microbes. Hence, the hypothesis was that the liberated methionine portion into the rumen following dietary RPM supplementation could affect the composition of major ruminal bacteria around calving.
Methods Animal handling and all procedures of this study received approval from the Institutional Animal Care and Use Committee of the University of Illinois under protocol no. 13023. In total, a subset of 20 multiparous cows from a larger study were randomly selected for ruminal fluid sampling. These cows were either fed a control diet without RPM (CON) or CON plus RPM supplementation (SM) at a rate of 0.08% of DMI (Smartamine M, Adisseo NA, Alpharetta, GA). Dosage of RPM was based on previous work from our research group . At the start of the experimental feeding phase, none of the cows enrolled had received any type of RPM. All cows received a far-off diet (Table 1 ) as total mixed ration (TMR) from -50 to -22 d before expected calving date (1.40 Mcal/kg of DM, 10.2% rumen-degradable protein, and 4.1% rumenundegradable protein), a close-up diet from -21 d to the expected calving date (1.52 Mcal/ kg of DM, 9.1% rumendegradable protein, and 5.4% rumen-undegradable protein) and a lactation diet after calving through 30 d postpartum (1.71 Mcal/kg of DM, 9.7% rumen-degradable protein, and 7.5% rumen-undegradable protein). The TMR was delivered once daily (0600 h) using an electronic recognition feeding system for each cow (American Calan, Northwood, NH) before calving or in open individual mangers during lactation. The DM content of the TMR for the close-up and lactation diets was measured weekly for estimation of daily TMR dry matter offered. The required amount of RPM was calculated daily for each individual cow and was top-dressed from -21 ± 2 to 30 d relative to parturition once daily at the morning feeding using approximately 50 g of ground corn as carrier. Ruminal bacteria DNA isolation and qPCR amplification of 16S rDNA genes At -10 d before expected calving date and d 20 postpartum, ruminal fluid was sampled from each cow using a stomach tube prior to the morning feeding. The sample was filtered through three layers of cheesecloth. Mixed ruminal fluid was immediately frozen in liquid nitrogen followed by storage at -80 C. Total genomic DNA was isolated using the repeated bead beating method described by Yu and Morrison for mechanical lysis of bacterial cell wall, employing the QIAamp DNA mini kit (QIAGEN) for DNA purification. The DNA quantity and quality were checked by 0.8% (wt/v) agarose gel electrophoresis and NanoDrop spectrophotometer (ND 1000, NanoDrop Technologies, Inc., Wilmington, DE, USA) at 260 nm. Extracted DNA was standardized to 8 ng/μL for qPCR. The primer sets selected to amplify 10 targeted rumen bacterial species are listed in Table 2 and were validated using gel electrophoresis. A total of 10 μL of qPCR mixture contained 4 μL sample DNA, 5 μL 1× SYBR Green with ROX (Quanta BioSciences, Gaithersburg, MD, USA), 0.4 μL each of 10 μmol/L forward and reverse primers, and 0.2 μL DNase/RNase free water in a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems, Foster City, CA, USA). Negative controls without template DNA, standards, and samples were run on the same plate in triplicate. The qPCR reactions were performed with the ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following program: initial denaturation at 95 C for 5 min, followed by 40 cycles of 1 s at 95 C and 30 s annealing at 60 C, except for eubacterial primer 3 that required an annealing temperature of 56 C. A dissociation stage was performed to determine the specificity of the amplification. Relative abundance of bacterial species was calculated using the geometric mean of two universal primers [19, 20] with the efficiency-corrected Δ -CT method . Thus, the abundance of each target bacteria was determined relative to the overall abundance of the total bacteria as measured with the universal primers.
Statistical analysis Dry matter intake and relative abundance of bacteria were analyzed using the MIXED procedure of SAS 9.3 (SAS Inst. Inc., Cary, NC, USA). The fixed effects in the model were Day, Treatment, and the interaction of Day × Treatment. The random effect was cow within treatment.
Results
Dry matter intake As expected, there was an increase in overall DMI after parturition (Day, P < 0.05) during which cows consumed on average 19.6 kg/d versus 13.9 kg/d in the prepartum period. The overall effect of treatment (P < 0.05) was due to cows in the SM group consuming ~3 kg DM more than those in the CON group specifically after parturition (Fig. 1 ; 19.6 kg/d versus 13.9 kg/d).
Abundance of ruminal bacteria The relative abundance of target bacterial species is presented in Table 3 . Selenomonas ruminantium was the only bacterial species with an overall treatment effect (P = 0.01) due to a 27.4% decrease in abundance with SM supplementation. Furthermore, this was the mostabundant bacterial species ranging from 2.63×10 -1 at -10 d (SM) to 6.39×10 -1 (a peak in abundance) at 20 d in cows fed CON. Concerning day effects, greater relative abundance of Fibrobacter succinogenes (P < 0.01) and Succinivibrio dextrinosolvens (P = 0.05) was observed at -10 d compared with 20 d postpartum. Furthermore, there was a day effect (P < 0.01) for Anaerovibrio lipolytica, Megasphaera elsdenii, Prevotella bryantii, Selenomonas ruminantium and Streptococcus bovis (P = 0.02) as these bacterial species had greater abundance at 20 d compared with -10 d The relative abundance of Butyrivibrio proteoclasticus and Eubacterium ruminantium was stable (P > 0.05) across treatment and time. However, Eubacterium ruminantium was the second most-abundant bacterial species among those studied, ranging from 1.92×10 -2 at -10 d with SM to 3.13×10 -2 at 20 d also with SM. A Day × Treatment interaction was observed for Fibrobacter succinogenes, Selenomonas ruminantium, Succinivibrio dextrinosolvens, and S. amylolytica. Selenomonas ruminantium abundance nearly doubled (P = 0.01) between -10 and 20 d only in CON cows. In contrast, Fibrobacter succinogenes, Succinimonas amylolytica and Succinivibrio dextrinosolvens decreased on d 20 in SM cows while no change or a decrease was observed in CON cows.
Discussion Selenomonas ruminantium was the most abundant bacterial species observed in the present study and agrees with previous classical studies indicating that this Gramnegative bacteria could account for up to 51% of the total viable bacterial counts within the rumen . The fact that Selenomonas ruminantium was overall 27.4% lower in response to RPM supplementation could have been associated with the greater DMI in those cows after parturition. This microbial species is a propionateproducer through decarboxylation of succinate, which involves lactic acid production particularly during feeding of higher-concentrate diets [23, 24] . Thus, because the numbers of Selenomonas ruminantium in SM-fed cows actually increased numerically after parturition relative to the prepartum period, it is likely that the greater DMI in those cows either diluted the numbers of this species or increased the diversity of the rumen population. Availability of propionate for gluconeogenesis by the animal during the transition into lactation relies heavily on both Selenomonas ruminantium and Megasphaera elsdenii numbers in the rumen . Approximately 90% of glucose in ruminants is supplied by gluconeogenesis, with 50 to 60% of this being derived from propionate . Thus, the longitudinal change in Selenomonas ruminantium and Megasphaera elsdenii agrees with higher content of rapidly-fermentable carbohydrate in the postpartum diet, i.e. substrate availability likely helped enhance the numbers of these species . Except for Butyrivibrio proteoclasticus (fibrolytic species), the greater numbers of Anaerovibrio lipolytica, Prevotella bryantii, Megasphaera elsdenii, Selenomonas ruminantium, and Streptococcus bovis at d 20 postpartum along with the lower numbers of Fibrobacter succinogenes, Succinimonas amylolytica, and Succinivibrio dextrinosolvens were consistent with a previous study . They attributed these changes to the important features associated with the transition into lactation, e.g. greater concentration of lipid and rapidly-fermentable carbohydrate as a way to provide more energy for the cow during a period when voluntary DMI may be less than optimal. The fact that abundance of Streptococcus bovis and Prevotella bryantii at 20 d postpartum was associated with greater abundance of Selenomonas ruminantium and Megasphaera elsdenii seems to indicate some degree of synchronization. These results are broadly consistent with other studies demonstrating that, as dietary grain increases, the prevalence of starch-fermenting bacteria like Streptococcus bovis is increased with a consequent A. lipolytica 3.28×10 -3 3.42×10 -3 2.50×10 -3b 4.48×10 -3a 2.25×10 -3 4.77×10 -3 2.77×10 -3 4.21×10 -3 0.82 <0.01 0.42 B. proteoclasticus 1.07×10 -2 8.88×10 -3 1.01×10 -2 9.46×10 -3 1.24×10 -2 9.29×10 -3 8.18×10 -3 9.64×10 -3 0.20 0.67 0.13 E. ruminantium 2.44×10 -2 2.45×10 -2 2.23×10 -2 2.69×10 -2 2.59×10 -2 2.31×10 -2 1.92×10 -2 3.13×10 -2 0.98 0.38 0.16 F. succinogenes 7.10×10 -4 6.93×10 -4 1.16×10 -3a 4.25×10 -4b 6.29×10 -4B 8.02×10 -4B 2.13×10 -3A 2.25×10 -4C 0.92 <0.01 <0.01 M. elsdenii 3.90×10 -5 1.90×10 -5 2.40×10 -6b 2.34×10 -4a 4.60×10 -6 2.07×10 -4 1.00×10 -6 2.65×10 -4 0.43 <0.01 0.24 P. bryantii 4.22×10 -3 1.30×10 -3 7.61×10 -4b 7.20×10 -3a 1.12×10 -3 1.60×10 -2 5.19×10 -4 3.25×10 -3 0.12 <0.01 0.57 S. ruminantium 4.10×10 -1a 2.98×10 -1b 2.63×10 -1b 4.65×10 -1a 2.63×10 -1C 6.39×10 -1A 2.63×10 -1C 3.37×10 -1BC 0.01 <0.01 0.01 S. amylolytica 1.02×10 -4 2.30×10 -4 2.51×10 -4 9.40×10 -5 9.90×10 -5B 1.05×10 -4B 6.32×10 -4A 8.40×10 -5B 0.16 0.07 0.05 S. dextrinosolvens 9.10×10 -5 1.60×10 -4 1.71×10 -4a 8.50×10 -5b 8.10×10 -5B 1.02×10 -4B 3.64×10 -4A 7.00×10 -5B 0.10 0.05 0.01 S. bovis 8.09×10 -3 3.50×10 -3 3.17×10 -3b 8.93×10 -3a 6.68×10 -3 9.79×10 -3 1.51×10 -3 8.14×10 -3 0.22 0.02 0.14 Means for the interaction of Day × Treatment differ (P < 0.05) synchronized increase in the population ratio of lactateconsuming bacteria like Selenomonas ruminantium and Megasphaera elsdenii to help eliminate lactate via fermentation to propionate [25, . Prevotella spp. grows rapidly on starch and produce succinate and propionate as major end-products [29, 30] . A previous study attributed a higher proportion of Megasphaera elsdenii and Prevotella bryantii after parturition to the higher DMI, which agrees with data in the present study. The lower abundance of Fibrobacter succinogenes postpartum was associated with greater abundance of Anaerovibrio lipolytica and seems to be partly explained by a potential negative effect of higher availability of dietary unsaturated fatty acids on ruminal cellulolytic bacteria . Indirectly, these results suggest that the greater fiber content of the prepartum diet allowed for greater numbers of Fibrobacter succinogenes, which agrees with a previous study . In ruminants fed high-forage diets Fibrobacter succinogenes is one of the predominant cellulolytic species, favoring the greater production of acetate relative to propionate . The greater abundance of fibrolytic bacteria such as Fibrobacter succinogenes, and starch-degrading bacteria such as Succinimonas amylolytica and Succinivibrio dextrinosolvens at -10 d relative to parturition in the SM group was not associated with differences in DMI; however, the lower abundance of these species in response to SM at 20 d could have been related with the greater DMI in those cows, potentially due to a change in the rumen kinetics such as liquid dilution rate. It is well-established that increases in dietary concentrate/grain to forage not only increase intake but also liquid dilution rate . Thus, besides the greater DMI in cows fed SM, the higher content of corn grain in the postpartum diet (Table 1 ) might have reduced rumen-retention time of digesta which could partly explain the lower abundance of bacteria. Recent studies demonstrated that RPM can increase DMI both pre and postpartum in dairy cows [16, 34] . It remains to be determined if the greater DMI and lower relative abundance due to feeding SM would affect digestive enzyme activity within the rumen. The response of Anaerovibrio lipolytica after parturition confirms its role in the hydrolysis of triacylglycerol into free fatty acids in the rumen . The inverse relationship between Anaerovibrio lipolytica and Fibrobacter succinogenes as it relates to dietary fiber and lipid level was confirmed previously . A previous study also speculated that Anaerovibrio lipolytica (like Megasphaera elsdenii) can use lactate as a substrate for growth during feeding of higher-fermentable diets after parturition. The lack of day, treatment, or interaction effect on the relative abundance of Butyrivibrio proteoclasticus and Eubacterium ruminantium implies that these bacteria might be less sensitive to changes during the transition period. Clearly, the diets used in the present study appear to have provided enough fermentable energy and nitrogen sources to allow normal growth of the major microbes studied. Whether the rumen microbiome responds to the supply of rumen-protected supplements needs to be explored further. This is particularly important because the actual requirements of methionine (and lysine, for example) during the transition period are unknown.
Conclusions In diets with proper balance of rumen-degradable protein and fermentable carbohydrate, the small fraction of Methionine released from the rumen-protected supplement did not seem to compromise growth of major bacterial species in the rumen. In fact, it had a positive effect on 3 major species prepartum when DMI was similar between groups. Because the actual requirements of Methionine (and Lysine, for example) by the cow during the transition period are unknown, it appears warranted to study the rumen microbiome as it relates to supply of rumen-protected amino acids. Fig. 1 1 Fig. 1 Dry matter intake in Holstein cows fed a control diet (CON) or CON supplemented with rumen-protected methionine (SM) from d -21 through d 30 relative to parturition. ab Different letters denote treatment effects (P < 0.05) between treatments. The P value for the overall effect of Treatment, Day, and Treatment × Day was 0.002, 0.03, and 0.48, respectively. Bars indicate standard error of the means
1 Trt treatment effect, Day day effect, T × D Treatment by Day interaction ab Means for overall Treatment or Day effect differ (P < 0.05) A-C
Table 1 1 Ingredients and chemical composition of experimental diets Diet Ingredient, Far-off Close-up Lactation % of DM Alfalfa silage 12.00 8.34 5.07 Alfalfa hay - 4.29 2.98 Corn silage 33.00 36.40 33.41 Wheat straw 36.00 15.63 2.98 Cottonseed - - 3.58 Wet brewers - 4.29 9.09 grains Ground shelled 4.00 12.86 23.87 corn Soy hulls 2.00 4.29 4.18 Soybean meal, 7.92 2.57 2.39 48% CP Expeller soybean meal a - 2.57 5.97 Soychlor b 0.15 3.86 - Blood meal, 85% 1.00 - - CP ProVAAl AADvantage c - 0.86 1.50 Urea 0.45 0.30 0.18 Rumen-inert fat d - - 1.02 Limestone 1.30 1.29 1.31 Salt 0.32 0.30 0.30 Dicalcium phosphate 0.12 0.18 0.30 Magnesium oxide 0.21 0.08 0.12 Magnesium sulfate 0.91 0.99 - Sodium bicarbonate - - 0.79 Potassium carbonate - - 0.30 Calcium sulfate - - 0.12 Mineral vitamin mix e 0.20 0.17 0.18 Vitamin A f 0.015 - - Vitamin D g 0.025 - - Vitamin E h 0.38 0.39 - Biotin - 0.35 0.35 a SoyPLUS (West Central Soy, Ralston, IA) b By West Central Soy c Perdue AgSolutions LLC (Ansonia, OH) d Energy Booster 100 (Milk Specialties Global, Eden Prairie, MN) e Contained a minimum of 5% Mg, 10% S, 7.5% K, 2.0% Fe, 3.0% Zn, 3.0% Mn, 5,000 mg of Cu/kg, 250 mg of I/kg, 40 mg of Co/kg, 150 mg of Se/kg, 2,200 kIU of vitamin A/kg, 660 kIU of vitamin D 3 /kg, and 7,700 IU of vitamin E/kg f Contained 30,000 kIU/kg g Contained 5,009 kIU/kg h Contained 44,000 kIU/kg
Table 2 2 Species-specific primers for the quantification of selected rumen bacterial populations using a real-time qPCR assay Target bacterial species a F forward primer; b R reverse primer
Table 3 3 Relative abundance of microbial species in mixed ruminal fluid from Holstein cows fed a control diet (CON) or CON supplemented with rumen-protected methionine (SM) during the periparturient period Day × Treatment
|
10.1111/imr.13191
|
cc-by
| null | null |
openalex
|
This article is part of a series of reviews covering Ontogenic Waves of Immune Cell Development appearing in Volume 315 of Immunological Reviews.| MA S T CELL ONTOG ENY Mast cells are tissue-resident innate effector cells of the myeloid lineage. They are critical for host defense against bacteria such as Staphylococcus aureus 1 and other pathogens like helminths. They also mediate protection from venoms produced by a range of animals, including snakes and bees. With this in mind, it is perhaps not surprising that mast cells are evolutionarily conserved. Cells with mast cell features are already present in tunicates, where they may have protective functions that are still relevant to modern mammals. They were first described by Paul Ehrlich in 1877 as cells filled with characteristic cytoplasmic granules. 11 These granules were thought of as either remnants of phagocytosis, or signs of mast cells feeding or nourishing surrounding cells. The latter interpretation gave them their name from the German word "mästen." Based on their localization and granule content, mast cells are typically classified as either mucosal (MTMCs) or connective tissue-type mast cells (CTMCs), which in humans, respectively, express tryptase or tryptase and chymase. Recognized already in the 1970s, there is now growing appreciation that mast cell heterogeneity extends beyond this dichotomy. For example, connective tissue-type mast cells share a core transcriptional program but differ between the skin, peritoneal cavity, and the esophagus, trachea, and tongue. 15 Moreover, mast cell phenotypes are not rigid, but can change in inflammatory conditions. Following their discovery, the original belief was that mast cells are of mesenchymal origin. 22 This notion was first challenged 40 years ago with the demonstration that bone marrow (BM) transplanted into irradiated hosts can generate mast cells. 23 Using "beige" mice that have distinctively large granules owing to a mutation, Kitamura, and colleagues were able to identify donorderived mast cells in recipient tissues. These findings were the first indication that mast cells are indeed hematopoietic in nature. This was confirmed in in vitro studies and parabiosis, an experimental approach in which two animals are surgically joined, resulting in a shared blood circulation. Based on these landmarking studies, the consensus became that mast cells originate from BM hematopoietic stem cells (HSCs). A similar BM-centric view has also been held for other immune cells like macrophages, which were long thought to be exclusively derived from monocytes, despite evidence to the contrary. However, it is now abundantly clear that many tissueresident immune cell lineages, including macrophages and certain innate lymphocytes, first originate from fetal-restricted progenitors. Over the life course, these fetal-derived cells can be supplemented with BM-derived ones. As we will discuss below, this developmental pattern also applies to mast cells.
| Layered hematopoiesis Hematopoiesis is a complex process that is initiated early in fetal development and involves several spatio-temporally distinct (but overlapping) progenitor waves (Figure 1 ). The first hematopoietic progenitors are produced in the extraembryonic yolk sac (YS). In the mouse, so-called primitive progenitors are made as early as day 7 of embryonic development (E7.0). These give rise primarily to erythrocytes and megakaryocytes, but also contribute to the very first macrophages. This is followed by the production of erythromyeloid progenitors (EMPs) starting at E8.25. 32, 33 EMPs can still differentiate into cells of erythroid, megakaryocytic lineages, but also more readily produce different types of myeloid cells. 33, 34 From E10.5 onwards, HSCs arise in the main arteries of the embryo properly. This occurs at several sites including the head, heart, placenta and vitelline, and umbilical veins, though most notably, the aorta-gonad-mesonephros (AGM) region. 43 Both, extra-and intraembryonic progenitors are generated from endothelial cells with hemogenic capacity in a process known as hemogenic-to-endothelial transition. 43 HSCs transiently colonize and are thought to expand in the fetal liver, although this view has recently been challenged 44 and hence, remains an area of ongoing debate. HSCs ultimately settle in the BM, where they persist lifelong. In addition to these welldescribed waves, there are other HSC-like progenitors generated (presumably) in the embryo proper starting at E9.0. 45, 46 These are less well-characterized, and since these waves overlap, their lineage output is difficult to disentangle from other progenitors. Alongside EMPs, these fetal HSCs are often referred to as transient-definitive. This is because unlike primitive progenitors, they express the transcription factor c-Myb, 47, 48 but they cease to exist perinatally and are thus transient in nature. To account for these differences, we here refer to EMPs and fetal HSCs as fetal-restricted, and to those HSCs that are produced in the AGM and take up residency in the BM as adult-type HSCs.
| Current understanding of mast cell origins As mentioned above, work published in the 1960s-1990s provided proof of concept that mast cells can originate from the BM. However, these seminal studies also already called into question whether the BM is the only source. It was observed that the rate of mast cell reconstitution from the BM is tissue-dependent, but overall low. Indeed, in the pioneering work from Kitamura et al 23 it took several months for the mast cell compartment along the gastrointestinal tract to be repopulated from grafted BM even in recipients conditioned by irradiation. Even after 6 months, very few if any donor BM-derived mast cells could be detected in the skin of these animals. Furthermore, mast cells with metachromatic granules can already be found in fetal tissues, that is, before the onset of BM hematopoiesis. 49, 50 In keeping with this, mast cell potential was identified in the extra-embryonic yolk sac and fetal liver. 27, 49, 51 To assess this, the Kitamura group adoptively transferred cells obtained from different fetal stages and sources into adult mast cell-deficient W/Wv mice (see Box 2), either intravenously or directly into the skin. 51 This revealed that between E9.5 and E11.5, the highest mast cell potential is found in the YS, followed by the fetal liver that predominates from E13.5 onwards. 51 The presence of progenitors with mast cell potential in the YS and fetal liver was also later confirmed in more refined assays. 33 Finally, work from the 1990 s described a population of lineage-committed cells present in the fetal blood. 52 ). EMPs differentiate into mast cells via committed progenitors that may seed peripheral tissues directly from the yolk sac and/or through fetal liver intermediates. The intra-embryonic aorta-gonad-mesonephros (AGM) region generates fetal-restricted hematopoietic stem cells (HSCs) between E9.0 and E10.0 and adult-type HSCs from E10.5 onwards. Both migrate to the fetal liver and may produce mast cells that supplement the first wave of EMP-derived mast cells. Around birth, adult-type HSCs colonize the bone marrow where they persist lifelong. Throughout life, bone marrow-derived progenitors further complement mast cells notably at mucosal sites and in conditions of environmental perturbations. Mast cell-committed progenitors express integrin α4β7, Cx3cr1, and Kit and undergo terminal maturation following recruitment into tissues. (Bottom) Origins of mast cells in adult tissues. In connective tissues like the skin and adipose tissues like the ones harboring the mammary glands, mast cells remain predominantly of fetal origins, with contribution from yolk sac EMPs and fetalrestricted HSCs. In the lung and gut mucosa, fetal-derived mast cells are largely replaced by bone marrow HSCs. undergone partial differentiation prior to reaching their destination tissue. Alternatively, this population may represent mature fetal mast cells that are transiently present in the fetal circulation, which are terminally differentiated, but phenotypically distinct from their adult counterparts. Collectively, these findings showed that mast cells can in principle originate from fetal-restricted progenitors found in the YS and fetal liver. However, they were based on either in vitro differentiation assays or adoptive transfer into hosts that were not age-matched and either pre-conditioned by irradiation or mast cell-deficient. It thus remained unclear whether fetal progenitors indeed produce mast cells under physiologic conditions, and if mast cells originating from such fetal-restricted progenitors persist postnatally. These questions were re-addressed recently using genetic fate mapping, which can establish lineage relationships between progenitors and their progeny. Three different fate mapping approaches unequivocally established that in vivo and under physiological conditions, the first mast cells are produced from YS EMPs. 53, 54 The frequency of EMP-derived mast cells gradually declines over time, although sizeable populations are retained in adult connective tissues, such as the body skin, as well as adipose tissues. Simultaneously, the frequency of labeled mast cells increases with age in models that capture later hematopoietic waves. This demonstrated that the first wave of EMP-derived mast cells is gradually diluted by mast cells originating from later progenitors. By far and large, the different models produced consistent data. However, different conclusions were drawn about the extent to which EMP-derived mast cells persist postnatally, and about which progenitors constitute the second mast cell wave. We believe that these apparent discrepancies can be explained by the specifics and inherent limitations of the various fate-mapping models used (discussed in Box 1). Consequently, we think existing data can be reconciled in an updated model of mast cell origins, whereby the first mast cells originate from a single wave of EMPs that are produced by the YS in a continuous manner (see Figure 2 ). These EMP-derived mast cells are then supplemented with a second wave that originates predominantly from fetal-restricted HSCs, but may also receive contribution from adult-type HSCs. Considering that turnover from the BM is very low for mast cells in connective tissues populations, 23, 53 such HSC-derived mast cell progenitors might be recruited in the perinatal window prior to settling in the BM.
| How do fetal progenitors generate mast cells? In mice, granule-containing, terminally differentiated mast cells can first be detected in the developing cornea at E12.5. 55 In contrast, mast cells loaded with heparin-containing granules have been reported only at E14.5-E15.5 in the murine and rat skin. 49, 53, 56 Moreover, others failed to detect them until E16.5 in mouse skin. 54 This indicates that mast cells populate different organs at distinct developmental stages. Kinetics may even vary slightly for distinct regions of the same organ, such as the skin, which could explain differences between studies. As we will discuss later, this may reflect that mast cell colonization is driven by tissue-and stage-specific demands, as has been discussed for macrophages. 57 Lastly, it is possible that fetal mast cells carry out effector functions even when they do not yet have granules. Indeed, mast cells found in mesenteric lymph nodes of adult mice infected with the helminth Trichinella spiralis potently produce IL-4 and IL-6 despite being hypogranulated. 58 During development, mast cells may thus be functional earlier than granule detection alone would suggest. In adulthood, progenitors first commit to the mast cell lineage and then undergo terminal maturation in peripheral tissues. Similarly, EMPs produce macrophage-committed progenitors ("premacrophages") that enter tissues via the blood stream and give rise to fetal macrophages. It is thus likely that the same applies to EMPs producing the first mast cells. Committed progenitors with an immature mast cell phenotype can be found in the circulation and peripheral tissues of developing mice and rats from E11.5 onwards. 54, 56 These do not yet have granules but express cardinal markers of mast cells such as Kit, as well as several mast cell proteases, at least on transcriptional level. 54, 59, 60 This indicates that lineage commitment indeed takes place prior to tissue colonization, while final maturation of fetal mast cells occurs in tissues. EMP-derived pre-macrophages populate the fetus in a chemokine-dependent process. 61 This is at least partially mediated by interactions between Cx3cl1 (also known as fractalkine) and its receptor Cx3cr1, 62 which is also expressed by fetal mast cells and their progenitors. 53, 63 The Cx3cl1/Cx3cr1 axis may thus also be involved in the trafficking of mast cell progenitors to peripheral tissues. This could be addressed in Cx3cr1 knockout mice. Considering that for macrophages, numbers in peripheral tissues are only transiently reduced early during development, and normalized later on, it will be important to also assess the relevance of this signaling axis for mast cells at the earliest stages possible, that is, when mast cells are normally detectable in substantial numbers in tissues. 62 Mast cell progenitor recruitment likely also involves integrin signaling. Integrin α4β7 specifically is expressed by adult BM progenitors with mast cell potential 64 and required for gut homing of mast cell progenitors. 65 Indeed, integrin α4β7 is expressed by EMP-derived progenitors that can be found in the fetal liver at E11.5. 53, 54 These progenitors phenotypically still resemble EMPs, 66 except for the lack of CD93 expression. 54 Following in utero transplantation into the liver of age-matched, unconditioned recipients, these EMP-derived integrin α4β7 + progenitors readily produce mast cells detectable in the skin and peritoneal cavity by E18.5. Phenotypically similar fetal liver progenitors that lack integrin α4β7, on the other hand, do not give rise to mast cells. These data provide proof of concept that EMP-derived fetal mast cell progenitors are marked by expression of integrin α4β7. However, integrin α4β7 is also expressed on progenitors for other immune cells, including BM-derived progenitors for T cells and innate lymphoid cells, and is often thought of as a more general tissue homing marker. 67, 68 The nature of the mast cell-specific committed progenitor downstream of EMPs (and potentially other Another open question is whether mast cell progenitors can directly seed fetal tissues, as is the case for the first macrophages, or always require a fetal liver intermediate. Based on granule staining, terminally differentiated mast cells may appear after macrophages. It thus seems plausible that EMPs will first pass through the fetal liver before committing to the mast cell fate. In support of this notion, progenitors with mast cell potential can be found in the fetal liver from E11.5 onwards, and these progenitors give rise to mast cells when transferred into fetal or newborn recipients. 54, 69 Nonetheless, this does not exclude that a direct route from the YS exists as well. Indeed, based on transplantation assays, mast cell potential is present in the YS until E13.5, and mast cells are in fact generated more efficiently from the YS than the fetal liver until E11.5. 51 At E11.5, EMP-derived mast cell progenitors expressing integrin α4β7 are also already present in the fetal circulation and body. 54 Interestingly, those found in the limbs are transcriptomically very similar to those in the fetal liver at the same stage, yet do show some differences in gene expression, suggesting they have either started differentiating in situ and must have been recruited earlier, and/or they may have originated elsewhere. To delineate these options, one would need to compare mast cell progenitors from the different hematopoietic sites and peripheral tissues at various developmental stages in a comprehensive manner. Taken together, existing data are in line with YS EMP-derived mast cell progenitors colonizing the developing fetus both directly and via the fetal liver.
BOX 1 What are the origins of the second mast cell wave? While several experimental strategies all identified yolk sac (YS) erythro-myeloid progenitors (EMPs) as the source of the first mast cells, different conclusions were drawn about the origin of the second wave. Based on Cdh5-CreERT2 fate mapping, we postulated that the second mast cell wave derives from adult-type HSCs, whereas Li and colleagues concluded from the Runx1-CreERT2 model that they originate from "late EMPs," with a minor contribution from "fetal HSCs." 54 To consolidate these interpretations, it is important to consider that hematopoietic waves overlap spatially and temporally (see Figure 2 ). YS EMPs, the much more ill-defined fetal-restricted HSCs and adult-type HSCs are all initiated in short succession between E8.25 and E10.5. However, each are produced for at least 1-2 days and generate immune cell output for even longer. None of the currently available models can spatially distinguish YS from intra-embryonic hematopoiesis, and all models available to study mast cell origins rely on a temporally defined distinction of different progenitors. Early, late, or one kind of EMPs? The Cdh5-CreERT2 and Runx1-CreERT2 models exploit that the genes driving Cre recombinase are required for all (definitive) hematopoietic waves. Both rely on 4-Hydroxy-tamoxifen (4OHT) for label induction, the active metabolite that allows more precise timing than tamoxifen. However, 4OHT remains bio-available for at least 24 hours. 246 There is thus substantial overlap between progenitors labeled, for example, in Runx1-CreERT2 mice pulsed at E7.5, E8.5, or E9.5. Moreover, the strict segregation of "early" and "late" EMPs (sometimes also referred to as EMP1 and EMP2) may simply reflect the timing of label induction, rather than biological differences. "Early" and "late" EMPs labeled in Runx1-CreERT2 mice upon induction at E7.5 or E8.5 might thus simply correspond to capturing the two extremes of one continuous YS EMP wave. Indeed, intrinsic differences between "early" and "late" EMPs have not been described to our knowledge. On the contrary, EMPs obtained from the YS and fetal liver at different stages behave the same in clonogenic assays in vitro. 32, 247 We thus think that rather than constituting a distinct second wave of "late EMP"-derived mast cells, these still represent the first wave that is generated from EMPs produced in a continuous fashion in the YS.
Fetal-restricted or adult-type HSCs? The question then remains where the second wave of mast cells comes from that gradually dilutes EMP-derived mast cells. Since bone marrow (BM) transplantation yields very limited mast cells even when transplanted into newborn mice, 54 adult-type HSCs either would need to seed tissues perinatally and bypass the BM, or they are not the major progenitor source. Both options seem plausible: Analysis of adult blood and immune cell lineages with known BM HSC origin confirms that induction of Cdh5-CreERT2 at E10.5 does label adult-type HSCs, 53 nonetheless, this approach may also label fetal-restricted HSCs. Conversely, E9.5 induction in the Runx1 model may not only capture fetal-restricted HSCs, but also some adult-type ones. High levels of neutrophil labeling in 4 weeks old mice indicate that this is indeed the case, since neutrophils are short-lived and of BM HSC origin at this stage. A very recent study supports a model whereby fetal-restricted HSCs are the main source of the second mast cell wave. From a combination of genetic fate mapping, adoptive transfer, and in vitro differentiation assays, Yoshimoto and colleagues conclude that only the earliest HSCs found in the fetal liver give rise to mast cells, but not those present at a time when adult-type HSCs dominate prior to BM colonization. 69 It would follow that mast cells are established from YS EMPs, followed by fetal-restricted HSCs. Adult-type HSCs may contribute in a brief perinatal window without passing through the BM. While mast cells in connective tissues largely self-maintain thereafter, mucosal mast cells receive further input from the BM after birth. Lastly, it is unclear which signals instruct fetal progenitors to commit to the mast cell lineage. They will likely involve stem cell factor (SCF) as well as IL-3 and key transcription factors like Gata2, which are needed for mast cell specification from BM progenitors, but may also include additional, fetal-specific factors. We also do not know how this core program is integrated with tissue-specific signals to define mast cell (sub)populations. For EMP-derived pre-macrophages, acquisition of tissue-specific transcriptional programs is initiated soon after their recruitment to peripheral tissues. This depends on unique transcription factors that are induced by micro-environmental cues. 61 Fetal mast cell specification may involve similar mechanisms. In line with this, human fetal mast cells show considerable heterogeneity across developmental stages (7-17 weeks of gestation) and tissues analyzed, that is, liver, BM, thymus, spleen, and skin. 60 These transcriptional differences parallel those seen in fetal macrophages. 60 Nonetheless, differences likely also exist. For example, (pre-) macrophages can be imprinted by environmental cues via phagocytic cargo, whereas mast cell progenitors or "pre-mast cells" may respond to different signals, and/or sense these in a different way.
| How are mast cells maintained postnatally? Decades of work have established that progenitors with mast cell differentiation potential exist in the adult BM. In both mice 64, 73 and humans, 74, 75 BM HSCs produce mast cells in a stepwise process from multipotent (MPP) to lineage-committed progenitors via common myeloid (CMP) and granulocyte-monocyte progenitor (GMP) intermediates. 76, 77 Within GMPs, bipotent progenitors (BMCPs) characterized by expression of Kit, CD34, CD16/32, and integrin α4β7 generate basophils and mast cells. Recent work that transcriptionally profiled BM progenitors on the single cell level found that BMCPs are segregated from GMPs by expression of FcεR1a and Ms4a2, which, respectively, encode the α and β subunits of the high-affinity IgE receptor, FcεR1. 78 Protein expression of FcεR1, on the other hand, appears to be variable for committed progenitors in peripheral tissues of mice and is also not found on human peripheral blood progenitors. 79 How basophil and mast cell fates are separated within BMCPs is still not fully resolved. Moreover, although the BM undoubtedly can produce mast cells, its actual contribution in vivo is limited under physiological F I G U R E 2 Waves of fetal hematopoiesis and their labeling using genetic fate mapping in mice. (Top) Overlapping waves of hematopoiesis during fetal development. Fetal hematopoiesis occurs in a rapid succession of several waves that partially overlap in space and time. The extra-embryonic yolk sac first produces primitive progenitors that do not contribute to mast cells. This is followed by single, continuous wave of erythro-myeloid progenitors (EMPs) that produce the first mast cells. At the time that EMPs production ceases, hematopoiesis commences intra-embryonically in the aorta-gonad-mesonephros region. The first hematopoietic stem cells (HSCs) are fetal-restricted but remain poorly defined. This is followed by the production of adult-type HSCs that colonize the bone marrow perinatally. (Bottom) Labeling of distinct and overlapping progenitor waves using genetic fate mapping. Key models that have been used to study mast cell ontogeny are shown. They depend on label induction in either endothelial cells with hemogenic activity (Cdh5, encoding vascular E-Cadherin), pan-hematopoietic progenitors (Runx1, encoding a transcription factor essential for hematopoiesis), or myeloid-committed progenitors (Csf1r, which encodes a growth factor receptor expressed by EMPs). Labeling is achieved through Cre-mediated recombination resulting in expression of a fluorescent protein. Recombination is induced at specific developmental time points matching hematopoietic waves through administration of 4-hydroxy-tamoxifen (4OHT). Double quoted annotations indicate what each labeling approach has been interpreted as in the literature on mast cell ontogeny. However, as discussed in the main text and Box 1, EMPs likely constitute a continuous wave (see main text and labeling partially overlaps between waves and fate-mapping models). This is illustrated by color-coded bars. Since there is currently no single fate-mapping model specific to either wave, data obtained in the different approaches should be interpreted in a complementary manner. | 37 CHIA et al. conditions. As introduced above, mast cell replenishment from BM transferred into lethally irradiated mice is restricted to mucosal sites like the cecum and stomach, in which approximately 50% of resident mast cells are of donor BM origin after 8 months. 23, 54 It has been suggested that this is due to tissue-specific differences in radiosensitivity of mast cells. Skin-resident mast cells, for example, are largely radio-resistant, 80 and there should thus be less need to replenish them from the BM. However, BM contribution is equally limited in "shielded" chimeras, in which most of the body is protected from irradiation, 53 as well as in parabiotic animals that are not irradiated at all. 24 Indeed, over a period of 4.5 months, only mast cells in the cecum, stomach, and spleen receive sizeable input from the parabiotic partner. 24 Radiosensitivity alone can thus not explain the strikingly different turnover rates observed for connective and mucosal mast cells. While cell-intrinsic difference may also contribute, environmental factors are likely key drivers of mast cell turnover. Mast cells residing in the intestinal mucosa, for example, are constantly exposed to food antigens and the microbiome. This could explain why they show the highest turnover from the BM, akin to intestinal macrophages. 81 Supporting this notion, mast cells are strongly reduced in the intestinal mucosa of germ-free mice. 82 Dermal mast cells, on the other hand, do not receive considerable BM input, despite also populating a barrier site. They are, however, also reduced and appear less mature in the skin of germ-free mice. 83 It will thus be critical to decipher exactly which factors drive maintenance and replenishment of mast cells at different sites, as well as their initial colonization during development.
BOX 2 Mouse models to study mast cell functions A wide range of mouse models is available to study mast cell functions in physiological and pathological settings. These either lack all mast cells, some populations or certain mast cell effectors. Kit mutant mice. Historically, mice were used that carry hypomorphic alleles of Kit, such as Kit W/Wv and Kit Wsh/Wsh mice. In these mice, mast cells are absent or reduced because they depend on Kit signaling for their growth and survival. However, Kit exerts pleiotropic functions both within and outside the hematopoietic system. 107, 248 As discussed by many others, these models are therefore by no means specific for mast cells. For example, Kit W/Wv mice have fewer neutrophils and basophils, are anemic and sterile. The Kit Wsh/Wsh model, on the other hand, is neither anemic nor sterile but has a higher number of neutrophils and basophils. Additionally, Kit-dependent models lack interstitial cells of Cajal and show abnormal pacemaker activity in the gastrointestinal tract. Results obtained from such models should be interpreted with caution, and their further use is discouraged. Kit-independent models for mast cell deficiency. Since the 2000s, the field has greatly benefited from the development of Kitindependent mouse models, many of which are based on the Cre/LoxP system. In these models, Cre recombinase is under the control of genes expressed (almost) specifically by mast cells. Genes coding for mast cell proteases are commonly used, such as Mcpt5 and Cpa3, the genes for mouse chymase and carboxypeptidase A3. Crossing these to the Cre-responsive strains Rosa26 lsl-DTA (for Diphtheria toxin alpha) or Rosa26 lsl-hDTR (for human Diphtheria toxin receptor), respectively, results in constitutive or inducible depletion of mast cells. 249 Alternatively, mast cell ablation can be achieved through Cre-mediated depletion of anti-apoptotic factors, such as Mcl-1. This is the principle underlying "Hello Kitty" mice (Cpa3-Cre; Mcl-1 fl/fl ). 250 Interestingly, an alternative approach to generating a Cre line using Cpa3 resulted in a model that constitutively depletes mast cells without further crossing. In these "Cre-Master" mice (Cpa3 Cre/+ ), knock-in into the endogenous Cpa3 locus results in Cre-mediated genotoxicity. 251 Other models were designed to achieve mast cell depletion in Cre-independent approaches. For example, conditional depletion of can be achieved using "Mas-TRECK" mice, in which DTR expression is under the control of intronic element of the IL-4 gene that normally drives IL-4 expression in mast cells. 252 In the "red mast cell and basophil" (RMB) mouse, DTR expression is under the control of the gene coding for the β chain of FcεR1 (Ms4a2), 253 allowing for inducible mast cell depletion and hence the study of their functions, for example, at different disease stages. Collectively, these Kit-independent models have allowed researchers to assess mast cell functions in a more specific manner. Indeed, using such models, many of the previous findings from Kit-dependent models have been challenged. 254 Nonetheless, these models are not without limitations. One main issue is that many of these also target basophils, albeit to varying degrees. Reduced basophil numbers are observed, for example, in Mas-TRECK mice. RMB mice are an interesting model in this regard, since basophils recover much more quickly from Diphtheria toxin-induced depletion than mast cells. This makes this model more mast cellspecific at later stages. Other potential issues include off-target effects or "Cre leakiness," which can in principle be a concern for all Cre-based approaches, as well as varying levels of depletion, certainly in inducible models. We therefore recommended using more than one model in a complementary manner where possible. Finally, most existing models do not differentiate (well) between (sub) populations of mast cells. While Mcpt5-Cre mice are useful to distinguish functions of connective-type from mucosal mast cell at least in the lung, 21 RMB, Mas-TRECK, and Cpa3-based mice are less restrictive. It will therefore be important for the field to develop new models that allow more subset-specific analysis of mast cells. An interesting explanation for the low degree of BM turnover has been put forward in the 1980-1990s, according to which mature mast cells may (actively) inhibit progenitor recruitment. 84, 85 This was proposed to explain why peritoneal mast cells are only replenished from donor BM when mature mast cells are absent. Potential mechanisms were not explored, but this model seems to be in keeping with a recent study. Using genetic complementation at the morula stage, Weitzmann and colleagues generated animals in which mast cells are labeled by either RFP or YFP expression under control of Mcpt5-Cre. 86 In this approach, (ear) skin mast cells form mono-colored clusters, indicating they have clonally expanded. At steady state, these clusters remain stable in size and distribution and are independent from BM input. 86 Moreover, mast cells do not invade adjacent clusters. Mast cell density and distribution are thus tightly regulated. While the underlying mechanisms remain unknown, the availability of growth factors will likely play a role. The key growth factor for mast cells is SCF, the ligand for KIT, which is hence also known as KIT-Ligand (KIT-L). SCF is required for mast cell (progenitor) chemotaxis, 87, 88 differentiation, adhesion, 89, 90 proliferation, and survival by blocking apoptosis. Mutations that render KIT constitutively active cause aberrant mast cell expansion, as we will discuss below. On the other hand, mice with hypomorphic alleles of Kit are profoundly deficient in mast cells and have therefore long been used as models to study their functions. However, SCF is also important for other cell types and developmental processes, including hematopoietic stem and progenitor cells, melanocytes as well as primordial germ cells and spermatogonia in the testis. Kit-dependent mice are therefore by no means mast cell-specific and findings obtained in these models should be interpreted with caution (see Box 2). SCF exists in soluble and membrane-bound forms, which are the result of alternative splicing of the same transcript. 94 The membrane-bound form is responsible for anchoring mast cells to the extracellular matrix, and it may also be the primary isoform required for mast cell development in vivo, since mice that have the soluble but not the membrane-bound form are mast cell-deficient. 95 SCF can be produced by a range of non-immune cells including fibroblasts and keratinocytes, and dermal mast cells are absent in mice in which SCF is specifically ablated in keratinocytes. 83 Mast cells may also produce SCF themselves, at least in vitro. 96 Exactly how SCF production is regulated remains unclear, but environmental factors like the microbiome likely play a role. Indeed, levels of SCF and consequently, mast cell numbers are lower in the skin of germ-free compared to conventionally housed mice. 83 While clearly important, availability of SCF and local mast cell density are unlikely the only factors which determine the extent and rate of their replenishment. Indeed, BM reconstitution is also surprisingly low even when recipients are used that are genetically mast cell-deficient, 97 in which it could be expected that the empty mast cell compartments are readily and rapidly repopulated from wild-type progenitors. Replenishment from grafted wild-type BM is not only very slow, it also does not restore wild-type numbers. 86, 97, 98 As these examples illustrate, we still do not fully understand which factors control mast cell numbers in healthy tissues.
| Non-homeostatic mast cell replenishment It has been proposed that BM progenitors are a primary source of mast cells following environmental perturbations, such as inflammatory insults. 99, 100 In an early publication, donor BM-derived mast cells appeared in the skin of lethally irradiated mice at sites that had been painted with methylcholanthrene, an irritant chemical with carcinogenic effects. 101 Substantial influx of BM progenitors to the skin also occurs following application of 12-O-tetradecanoylphorb ol-13-acetate (TPA), a small molecule drug originally derived from a plant that induces skin rashes. 86 Within TPA-treated inflamed skin, BM progenitors clonally expand and invade the territories of preexisting mast cells. 86 Similarly, Dwyer and colleagues identified a population of progenitor-like immature mast cells in nasal polyps of patients with chronic rhinosinusitis. 102 These are distinct from circulating mast cell progenitors and appear highly proliferative. Cells with similar features were also found in fibrotic and asthmatic lung tissue. 102 Finally, mast cell progenitors are also recruited to the airway mucosa upon allergic sensitization. 103 These are thought to be BM-derived, although this was not formally shown. Indeed, mast cell progenitors are also present in the spleen 104 and murine adipose tissues, 105 to which the latter are mobilized upon experimental myocardial infarction. 106 As in mice, committed mast cell progenitors (or immature mast cells) have also been found at extra-medullary sites in humans, including the spleen and a range of non-lymphoid tissues, and they too can be recruited in inflammatory conditions. 10 Overall, available data thus indicate that following perturbations,
| IMPLI C ATI ON S OF MA S T CELL ONTOG ENY FOR LIFE-LONG HE ALTH AND DISE A SE Mast cells are best known for mediating allergy and anaphylaxis, but they also have other pathological roles. Aberrant expansion or activation of mast cells causes conditions known as mastocytosis and mast cell activation syndrome, and mast cells are also found associated with tumors. On the other hand, mast cells can also be protective, for example against Staphylococcus, honeybee, and snake venom. 1, Moreover, they have also been implicated in a variety of non-immunological processes. These range from metabolism and neuronal crosstalk to tissue remodeling, wound healing, and angiogenesis, 107 conditions that are biologically reminiscent of development. Here, we discuss how their ontogeny may be relevant to healthy development and pathology.
| (Potential) functions of mast cells in early life Fetal mast cells share their YS EMP origin with macrophages, which are known to have pivotal roles in development. 31, 32, 48 Rather than waiting for adult-type HSCs that become the dominant source of immune cells perinatally, fetal tissues are thus seeded with mast cells from the earliest possible source, suggesting they are required before birth. Indeed, mast cells appear to be functionally mature at fetal stages: In mice, mast cells detected as early as E12.5 in the cornea and E14.5 in the skin already contain characteristic granules. 53, 55 At least on transcript level, fetal mast cells also express key effectors like proteases. 53, 54, 63 This is also true for human fetal mast cells, which express, for example, TPSAB1, the gene encoding tryptase, and carry granules. 59, 60, 63 While surface expression of FcεR1 is only upregulated perinatally, 53, 63 fetal mast cells express other receptors through which they can sense the environment, such as the IL-33 receptor. 53, 54, 63 They can also respond to environmental signals and stressors already in utero. 63 Producing functional mast cells at fetal stages may therefore serve at least two different purposes: Fetal mast cells could help protect from immunological threats before the adaptive immune system is set up, and/or they may support developmental processes.
| Do mast cells contribute to protective immunity at pre-and early postnatal stages? In adults, mast cells have important functions in protective immunity from certain bacteria, 1 helminths 2-4 and animal venoms. 6, 7 They are potent immune effectors that can rapidly respond to harmful stimuli owing to their intracellular granules loaded with pre-formed effectors. Furthermore, mast cells precede the adaptive immune system both ontogenetically and phylogenetically. It seems plausible therefore that mast cells are also important for protective immunity in early life. One hypothesis is that they are present prior to birth primarily to prepare newborn mammals for immunological threats, for example from venomous animals or helminths. Specifically, it has been proposed that fetal mast cells may be primed in utero, for example through maternal IgE, allowing stronger and faster responses to harmful stimuli encountered after birth. 10, 63, 108 If they were indeed critical for protective immunity in early life, it could be reasoned that prevalent infections constitute a strong evolutionary driver for maintaining mast cells. Helminth infections, for example, affect approximately one-third of the total population, with strong regional bias towards tropic regions. 109 Helminth infections are not commonly thought to be vertically transmitted to the fetus, however, transplacental infection has been described. 110 Moreover, transmission through the mammary glands is a major route of infection after birth. 111 Even where they are maternally restricted, helminth infections could be sensed by the developing fetus, for example by maternal cytokines. 112 This is evidenced by a growing body of literature reporting protective effects of maternal helminth infections on offspring susceptibility to allergic disease. While seemingly at odds with potentiating postnatal immunity, the effects of maternal infections appear to be dependent on the phase of infection experienced during pregnancy, which for example for Schistosoma mansoni ranges from a predominantly type I and II to a regulatory response. 112 It is thus conceivable that in utero priming of fetal (-derived) mast cells by maternal helminth infections can indeed potentiate anti-helminth immunity in infancy. Such a mechanism could be important considering that protective immunity against helminths is normally slow to develop. 109 In addition to preparing for potential threats after birth, fetal mast cells may already have protective immune functions in utero. This might be of particular relevance for infections that can cause developmental defects, preterm delivery, or stillbirth, such as Group B Streptococcus. 113, 114 Recent work demonstrated that mast cells help defend against these bacteria through the coagulation factor XIIIA. 115 It seems possible that fetal mast cells may complement the action of the maternal immune system, and thereby safeguard fetal development. Although there is currently no experimental data showing this directly, human fetal mast cells acquire transcriptional signatures compatible with immune effector functions already from 10 weeks of gestation onwards. 60, 116 Future studies should thus address if fetal mast cells help protect from maternally transmitted infections.
| Do mast cells support developmental processes? Threats from parasites, other infectious agents or venomous animals may be common at least in wild animals and some human environments. Yet, one could argue that challenges intrinsic to development affect all individuals of a population. If fetal mast cells supported developmental processes to overcome such challenges, then these would be powerful evolutionary drivers for maintaining MCs in early life. Although the idea that mast cells support developmental processes has gained traction, 10, 11, 60, 117 mice that lack mast cells develop normally without obvious defects, and mast cell deficiencies have not been reported in humans. The current consensus therefore is that mast cells are dispensable for development. Nonetheless, there is some evidence supporting developmental roles for mast cells. In rodents, they have been implicated in vasculature and nerve branching of the developing cornea. 55 Mast cells colonize the developing cornea in two waves. The first mast cells concentrate around the corneal stroma from E12.5 onwards and disappear by the time of eyelid opening. A second wave of mast cells settles in the corneal limbus within the first postnatal week and stabilizes thereafter. These are recruited to the corneal limbus via a mechanism that depends on integrin α4β7 and Cxcr2. 55 These kinetics mirror those of mast cells in other tissues, suggesting corneal mast cells may have similar developmental origins. At both stages, expansion of the local network to the required density may be achieved through in situ proliferation of mature mast cells. Of note, the area covered by blood vessels and the density of nerve fibers are both significantly reduced in newborn Kit Wsh/Wsh mice that do not have corneal mast cells. 55 This indicates that fetal mast cells mediate vascular and nerve branching in the developing cornea. The underlying mechanisms have not been explored in detail, but it was reported that corneal mast cells produce vascular endothelial growth factor (VEGF) and neurturin, which, respectively, have angiogenic and neurotrophic actions. In agreement with this, inappropriately high levels of mast cell-derived VEGF promote neovascularization in the adult cornea, that is, excessive angiogenesis that can lead to impaired or lost vision. 118 Mast cells can also be found in the developing brain of mice 54 and rats, 119 including the pre-optic area. 120 This brain region is essential for male copulatory behavior, which is marked by a higher synaptic density compared to females. Late fetal and early postnatal stages are critical for sexual differentiation in this region. At these stages, mast cell numbers are higher in the pre-optic area of males, where mast cells are actively degranulating in response to estradiol released due to a surge in testicular hormones. 120 Pharmacological inhibition of mast cells at late fetal stages dampens copulation behavior in adult male offspring, suggesting that masculinization is normally mediated by mast cells. Conversely, perinatal administration of exogenous estradiol or mast cell activation using the compound 48/80 both partially masculinize the copulation behavior of female mice. Mechanistically, mast cells modulate synaptic patterning indirectly through crosstalk with microglia. 120 Mast cells thus regulate sexual differentiation in the brain during development in a hormoneresponsive manner and via mechanisms that involve other immune cells. Whether they also contribute to other developmental processes in the brain remains to be explored. Developmental roles for mast cells may also extend beyond the fetal period, since many tissue maturation or remodeling processes continue or take place after birth. For example, female organs associated with reproduction undergo substantial tissue remodeling in preparation for pregnancy. In many aspects, the underlying biology of such tissue remodeling closely resembles fetal organogenesis. In the resting, non-pregnant uterus, the blood vessels are thick-walled. Early during pregnancy, these are converted into thin-walled vessels with large lumens. 121 This allows for effective exchange of oxygen and nutrients to meet the increased demands to the developing fetus. Of the innate immune cells that reside in the female reproductive organs, mast cells seem to be of particular importance for reproductive success. In the uterus, mast cell numbers increase during the receptive stage of the estrous cycle and early pregnancy, when they are in close association with the blood vessels at the implantation sites. 122, 123 Mast cell-deficient female mice display defective spiral artery remodeling and impaired implantation, collectively resulting in fetal growth restriction. 122, 124, 125 Interestingly, mast cells appear to mediate these processes cooperatively with uterine NK cells, since both can (partially) compensate in the absence of the other. 125 This functional redundancy may be a safety measure ensuring appropriate uterine remodeling. Mast cells have also been implicated in mammary gland remodeling at key developmental and reproductive stages. Mammary gland development is initiated at E10.5 in mice, but there are only a few rudimentary branches at birth. The glands remain relatively quiescent until puberty, when the ducts undergo rapid proliferation and branching, and epithelial cells form terminal end buds at the end of rudimentary ducts. Cells from these terminal end buds then invade the surrounding adipose tissue and form branches until the ductal tree is completed. During pregnancy, branching continues and ductal epithelium proliferates and differentiates to ultimately form the milk-secreting alveoli. Weaning then triggers mammary gland involution and return to the pre-pregnancy state. Mast cells localize to the terminal end buds of the mammary gland ducts during puberty, lactation, and subsequent involution. 126, 127 Mast cell-deficient Kit Wsh/Wsh mice show a delay in pubertal ductal branching. 128 Whether the transient nature of this defect is due to functional compensation by other cells is unknown. Likewise, the mechanisms by which mast cells may support branching morphogenesis in the mammary gland are not fully understood, but IL-33 signaling, and mast cell proteases are likely involved. The plasminogen cascade and the signaling axis between IL-33 and its receptor ST2 have been associated with both normal mammary gland development and tumorigenesis, 129, 130 and IL-33 signaling is important for mast cell survival and activation. 131, 132 In the pubertal and post-lactational mammary gland, mast cells also produce the protease plasma kallikrein, which activates the plasminogen cascade. 127 Mast cell proteases may also enhance IL-33 signaling 133 and promote stromal remodeling via secretion of pro-inflammatory cytokines like IL-6 134, 135 and IL-13. 136, 137 It is thus possible that effectors released by mast cells directly affect mammary gland remodeling, independently of other immune cells. 128 Finally, single cell transcriptional profiling recently demonstrated that the earliest mast cells found in human fetuses express transcripts for pro-inflammatory cytokines and chemokines involved in recruitment of endothelial cells. 60 This has been interpretated as an indication that fetal mast cells are involved in tissue morphogenesis or angiogenesis also in humans. 60 Although there is currently no further evidence for this, it has previously been speculated that human mast cell deficiencies may be embryonically lethal, 107 a hypothesis that we think should be considered. In conclusion, there is some evidence that mast cells do support healthy development at pre-and postnatal stages. Future studies should address if fetal(-derived) mast cells are important for protective immunity before and shortly after birth. Moreover, we believe that evolutionary and developmental considerations provide a strong rationale to explore if and how mast cells support developmental processes more broadly. As the examples of the cornea, brain, and mammary gland illustrate, mast cells are likely involved in fine-tuning such processes. The consequences of lacking mast cells may therefore be difficult to detect, in keeping with the overall normal appearance of mast cell-deficient mice and the apparent absence of (symptomatic) human mast cell deficiencies. Moreover, even where mast cells did normally support tissue maturation, other cell types may be able to compensate in their absence. As suggested for the mammary gland, mast cell deficiency may thus result in subtle, transient phenotypes or developmental delays, rather than persisting overt defects. Addressing their roles in normal development will thus require hypothesis-driven, in-depth analyses of kit-independent mast cell-deficient models (see Box 2) at key stages relevant to the tissue of interest. Models allowing stage-specific ablation may further aid in this. With such carefully designed approaches, we may ultimately be able to assess the relevance of findings obtained in mouse models for human development.
| Abnormal mast cell development and pathology Instead of supporting healthy development, mast cells can also become pathological. They can either rendered pathological themselves, through mutations or environmental programming, or their normal functions may be co-opted for example by the tumor microenvironment. In the following, we discuss how mast cell ontogeny may be relevant to pathology.
| Mastocytosis and mast cell activation syndrome Mastocytosis refers to a group of rare, heterogeneous disorders characterized by aberrant, clonal expansion of mast cells, which are accordingly classified as myeloproliferative neoplasms. Most cases are benign, but mastocytosis can also be associated with other hematological malignancies, 138 or can itself become malignant. Symptoms are complex and reflect the broad spectrum of biological activities of the released mast cell effectors. They range from skin reactions like itching, an increased risk of anaphylaxis and general fatigue to gastrointestinal complications such as cramps, diarrhea, and nausea as well as pain in muscles and joints. Different forms of mastocytosis are distinguished according to the affected tissues as well the age of onset and clinical course. In cutaneous forms, mast cell expansion is restricted to the skin. The first case of mastocytosis described as early as 1869 was Urticaria pigmentosa, 139 a cutaneous form, although it was only recognized as a mast cell disease several years. 140 In addition to the skin, systemic mastocytosis often also involves the BM and additional sited including lymphoid organs like spleen and lymph nodes and other internal organs like the gastrointestinal tract and liver. In some cases, symptoms associated with systemic disease like gastrointestinal cramping and anaphylaxis occur without apparent involvement of the BM. 141 Cutaneous and systemic mastocytosis are further distinguished from mast cell malignancies. 138, 142, 143 Different types of mastocytosis also show a remarkable distinct clinical course: Pediatric-onset disease is predominantly cutaneous, tends to follow a milder clinical course and usually regresses spontaneously by adolescence. Adultonset disease, however, is more frequently systemic and does not normally regress. While most adult patients have stable disease with different severities, adult mastocytosis can also turn into more aggressive, systemic disease, and even malignancy, 144, 145 which both have poor prognosis. 146, 147 Although attempts at a better classification are made regularly, 148 mastocytosis remains clinically complex, and curative treatments are currently lacking. There is thus currently an urgent clinical need to improve patient stratification and explore new therapeutic avenues. The cause of mastocytosis is mutational hyperactivation of KIT, the tyrosine kinase receptor for stem cell factor, which mast cell development and survival depend on. These mutations are typically somatic, and germline mutations are very rare. They result in constitutive or ligand-independent KIT signaling, leading to uncontrolled mast cell expansion. Auto-activating KIT mutations are a shared feature of different types of mastocytosis. 156 Most common is a substituting point mutation in codon 186 (D186V), which is found in 80% of adult patients. 157 Although less abundant in these patients, this mutation still accounts for almost 40% of pediatric cases, and of the remaining pediatric patients, another 40% carry activating mutations in other regions of the gene. 156, 157 This indicates that while the specific nature of the mutations may have some prognostic relevance, different mutation unlikely explain the distinct clinical courses of transient pediatric and chronic adult disease. Indeed, progress in our understanding of disease etiology on both, the cellular and molecular level notwithstanding, we still do not understand the biological differences underlying these striking differences. One aspect that has received limited attention is the possibility that pediatric-and adult-onset mastocytosis may have different cells of origin, specifically EMP-like progenitors and HSCs. Indeed, persistence of adult-and spontaneous regression of pediatric-onset disease resemble the normal kinetics of mast cell development that we have outlined above: In mice, the first, YS EMP-derived mast cells are gradually replaced by mast cells originating from later progenitor waves. 53, 54 These changes mainly occur from late fetal to early postnatal stages, which correspond to the time pediatric-and adult-onset are clinically distinguished, and the age regression often occurs in pediatric-onset patients. Intriguingly, activating KIT mutations have been described in HSCs and more committed downstream progenitors in the BM and peripheral blood of adult patients with systemic disease, 77, but not pediatric patients. This supports the hypothesis that pediatric-and adult-onset mastocytosis may have distinct cells of origin. To test this notion experimentally in mice, activating KIT mutations could be introduced into different mast cell progenitors using Cre recombinase technology, an approach that causes mastocytosis in vivo. 162 Another condition caused by aberrant mast cell responses is known as mast cell activation syndrome (MCAS). This heterogeneous disease is characterized by recurring episodes of allergic and anaphylactic symptoms, which typically involve multiple organ systems, such as the skin, lung, heart, and gastrointestinal tract. Common symptoms include skin itching, swelling, and flushing, wheezing, abdominal pain, cramping, and diarrhea. These symptoms are driven by high levels of potent mast cell mediators, 163 such as tryptase, histamine, leukotriene E4, and prostaglandin D2. 164 MCAS can present in combination with mastocytosis and other mast cell-related comorbidities such as Ehlers Danlos Syndrome, a rare genetic condition affecting connective tissues. Most cases of MCAS are idiopathic, 164 however. Symptomatic MCAS episodes appear to be triggered by environmental exposures, for example to psychological stress, 165 sudden temperature changes or odors and chemicals. 166 In addition to triggering symptoms, environmental exposures may also contribute to MCAS etiology: A recent study has explored a possible link between MCAS and (acquired) chemical intolerance, and proposed that xenobiotic exposures also render mast cells hyper-sensitive in the first place. 166 This study suggested that mast cell sensitization may be the result of either isolated high-dose exposures, or cumulative low-grade exposure events. Since mast cells are long-lived, both scenarios could induce long-lasting changes, for example, through epigenetic mechanisms, thereby causing MCAS. As mast cells are already present and responsive from fetal stages onwards, these considerations may extend to the pre-and early postnatal period. Indeed, MCAS can also affect children, and it is therefore tempting to speculate that environmental exposures experienced in utero or shortly after birth contribute to MCAS etiology. Keeping in mind the normal developmental kinetics of mast cells outlined above, it will be important to delineate the relative contribution of fetal-derived and adult mast cells to MCAS pathogenesis, as well as to decipher to what extent the underlying biology is shared or distinct.
| Mast cells as targets of developmental programming Based on numerous epidemiological studies, it is now firmly established that adult health and disease have developmental origins. 167 Exposure to adverse environments in utero or at early postnatal stages is associated with a wide range of non-communicable diseases, 168 many of which are characterized by inflammation and aberrant immune responses. 169 Maternal infections and inflammation, psychosocial stress, nutrition, pollution, and chemical exposures are among the many early life stressors shown to increase the risk of developing such pathologies. This phenomenon is known as pathological or developmental programming. Their ontogeny may make mast cells particularly susceptible to developmental programming: They are already present and phenotypically plastic at fetal stages and can sense environmental perturbations. 63, 170 They are also long-lived and many fetal-derived mast cells persist postnatally. Much like macrophages, 57, 171 with which they share developmental origins, mast cells may thus be functionally altered by exposure to early life adversity and thereby mediate or worsen disease in later life. Allergy is a cardinal mast cell-mediated pathology with established roots in early life. Allergic symptoms are caused by aberrant activation and degranulation of mast cells in response to otherwise innocuous stimuli and range from localized swelling and itching to life-threatening anaphylaxis. 172 The incidence of allergy is on the rise, both in developed and developing countries. Yet, while the central role of mast cells in driving pathology has long been established, 172 the etiology of allergy remains unclear. Sensitization to allergens may begin before birth. Indeed, heredity of allergy is more strongly associated with maternal IgE compared to paternal IgE. 180, 181 A recent study provided a biological basis of this phenomenon with the demonstration that fetal mast cells are sensitized by allergen-specific maternal IgE that passes the placenta, resulting in hypersensitivity upon postnatal encounter with the allergen. 63 This mechanism thus propagates maternal disease to the next generation and could explain the rapidly rising incidence of allergic disease. Additional stressors may also play a role in programming allergy in offspring. The rise in allergy incidence has also been associated with a reduced exposure to microorganisms in early childhood. 182 In what is known as the hygiene hypothesis, it was postulated that reduced microbial exposure results in a functionally restricted, "inexperienced" immune system that may be biased towards type II responses. 183 This view may be oversimplified, however, and it originally did not take into account the prenatal period, although prenatal exposure to microorganisms inversely correlates with the risk of developing atopy. 184 It has since been proposed that restricted maternal microbial exposure also renders their offspring hypersensitive, and that this is at least in part mediated by effects on fetal immune cells or their progenitors. 185 In keeping with the hygiene hypothesis, germ-free mice have reduced mast cell numbers in the intestinal mucosa and are resistant to food allergy models. 82 Future studies need to determine exactly how mast cells are shaped by fetal and newborn microbial exposure. They should also explore if and how mast cells are developmentally programmed by other stressors associated with allergy, such as pollutants, 186 nutrition and psychosocial distress. 190 A firm link with early life adversity has also been established for neuro-developmental and psychiatric disorders, 191 and immune dysregulation is involved in driving these pathologies. 192, 193 Central nervous system mast cells may be involved. Indeed, mast cells have been linked to anxiety, 194 and MCAS patients frequently report suffering from psychiatric symptoms. 195, 196 As discussed above, MCAS may be triggered by cumulative exposures to environmental triggers and may hence represent another example of mast cell programming. Hyperactivation of meningeal mast cells and behavioral changes were also reported upon maternal separation, an animal model for early life stress. Specifically, maternal separation results in reduced sucrose preference, a symptom of depression in rodents. 197 These effects are more pronounced in females than in males. 197 Stress-associated hyperactivation of mast cells may also play a role in neurodegenerative diseases. 198, 199 Lastly, mast cells are also associated with the peripheral nervous system, for example in the gut. 200 Early life adversity 201 and mast cell-neuronal interactions have been proposed as drivers of abdominal pathogenesis in gastrointestinal diseases like irritable bowel syndrome (IBS). 200, 202 The combination of maternal separation and early weaning induces symptoms of IBS in pigs, and these correlate with increased numbers and activation of intestinal mast cells. 203 Intestinal mast cells are also increased and hyperactivated in mice following maternal separation, and myenteric glia displayed a heightened responsiveness to histamine. 204 Mast cell hyperactivation by stress is likely at least in part driven by the corticotrophin-releasing hormone (CRH) axis that regulates physiological stress responses. 205 Moreover, aberrant placental CRH signaling has been liked to neurodevelopmental alterations in the fetus, 206 which could be driven by mast cells. Indeed, mast cells express receptors for CRH 207, 208 that may act in an antagonistic fashion. Signaling via CRH1 receptors triggers mast cell degranulation, 205 whereas signaling through CRH2 receptors does not. 209 Mast cell hyperactivation may thus for example be mediated by a disbalance in CRH receptors, which could be epigenetically imprinted. Microglia are developmentally programmed by prenatal stress in this way, 210 and similar mechanisms could thus apply to mast cells.
| Could ontogeny be the key to understanding the roles of mast cells in cancer? Mast cells were first found associated with carcinoma by Paul Ehrlich. 211 They are present in most solid tumors, where they may shape the tumor microenvironment and determine therapy response like other immune cells. Mast cells can affect tumor growth in several ways: They can promote angiogenesis through production of VEGF and Fibroblast Growth Factor 2 (FGF2), and indirectly through degradation of the extracellular matrix, which results in release of additional pro-angiogenic factors. 212 Matrix degradation also supports malignant tissue remodeling and tumor invasiveness. 213 Mast cells can also directly impact malignant cells. For example, mast cellderived histamine and TNFα regulate cytokine production by tumor cells, and tryptase can inhibit melanoma proliferation. 214 Finally, mast cells can promote either an immuno-suppressive tumor environment 215 or potent anti-tumor immunity, for example by recruiting cytotoxic lymphocytes. 216 Nonetheless, their roles in cancer have remained heavily debated. In clinical and experimental studies alike, mast cells have been associated with both, beneficial and detrimental outcomes. This is true often even for the same tumor types, including pancreatic, 217, 218 lung, 219, 220 colorectal 221, 222 and prostate cancer as well as melanoma. 220, Other reports concluded they have no role at all. The literature on melanoma is particularly controversial. Protumorigenic roles for mast cells were reported in studies from the early 2000 s, which found a correlation between mast cell counts and poor prognosis. 234, 235 These studies suggested that mast cells promote tumor angiogenesis through VEGF. Other studies reported anti-tumorigenic roles for mast cells in melanoma. For example, one report found that low densities of tryptase-and chymase-positive mast cells correlate with poor prognosis and a higher melanoma grade. 236 The authors suggested that high concentrations of serine proteases in the tumor microenvironment might impair melanoma growth. Indeed, it was later shown that melanoma cells can take up tryptase via exosome trafficking. 214 This led to cleavage of histone H3 and lamin B1 and extensive nuclear remodeling, ultimately leading to reduced tumor growth. Of note, cleavage of histone H3 and loss of lamin B1 also occur during cellular senescence, a process known to limit tumor progression. Finally, several mouse studies have found that the absence of mast cells has no effect on skin carcinogenesis or the growth of subcutaneously inoculated melanoma. They have thus concluded that mast cells are not essential for either melanoma growth or anti-melanoma immunity, and that mast cells are rather inert "bystanders." This interpretation remains difficult to reconcile with their (peri)tumoral enrichment, however, which suggests active recruitment. An alternative explanation is that melanoma-associated mast cells comprise different populations with distinct and potentially antagonistic effects on tumor growth. Although the Cre/LoxP-based models used in these studies represent a major improvement over traditional Kit-dependent models (see Box 2), many target mast cells rather indifferently.
Indeed, subcutaneous growth of B16 melanoma is unaffected in Mcpt5-Cre:Rosa DTA mice, 231, 232 which lack all mast cells in the skin. Intriguingly however, B16 metastases are drastically reduced in the lung, 232 where only connective-type mast cells are absent, but the mucosal-type population marked by expression of integrin α4β7 is retained. 21 This highlights how taking the heterogeneity of tumorassociated mast cells into consideration could unravel biological functions that may otherwise be masked. A key determinant of the heterogeneous mast cell functions in the tumor microenvironment may be their ontogeny. Again, much can be learned from the comparison with what we know about macrophages in tumors. Macrophages are recruited either de novo from BM-derived monocytes or as pre-existing mature cells from surrounding tissues, which again can have different origins. Fetalderived macrophages are often recruited to tumors at early stages 240 to promote their growth and metastatic spread. 243 They can also selectively interact with stromal cells that have reactivated fetal features, generating an "onco-fetal ecosystem" characterized by marked immuno-suppression. 244 Specifically, in hepatocellular Associated Protein (PLVAP), a marker of fetal liver endothelium. 244 Similar mechanisms may be at play in other tumor types. 244, 245 Based on their shared ontogeny, it is thus tempting to speculate that similar considerations also apply to tumor-associated mast cells. Future studies will need to address if they are recruited to tumors from different sources and at distinct stages, and whether such populations impact tumors in different ways.
| OPEN QUE S TI ON S AND FUTURE DIREC TIONS Our insight into mast cell biology is ever evolving. Advances in mouse models not only helped clarify their roles in a range of pathologies but also enabled the discovery of previously unappreciated mast cell functions. Through deep phenotyping, (single cell) transcriptomics, and proteomics, it has become abundantly clear that these cells are more heterogeneous than what is captured by the classical dichotomy of mucosal-and connective tissue-type mast cells. These approaches have also enabled studying human mast cells with unprecedented resolution. We also now have an overhauled understanding of mast cell development. Mast cells are already present in the fetus, where they can sense and respond to environmental signals and thus, appear functionally mature. They are long-lived, and most mast cell populations found in healthy connective tissues are established in the pre-or early postnatal periods and subsequently self-maintain with site-specific, but overall low input from the BM. These developmental dynamics have far-reaching implications for mast cell biology in health and disease (see Figure 3 ): Their presence at early life stages suggests that mast cells may be needed during development. Their early life origins and longevity also make mast cells highly susceptible to genetic and environmental insults, which may render them pathological. Much like for macrophages, mast cell identity, and functions are thus likely shaped by their ontogeny, that is, the combination of their cellular sources and differentiation trajectories, the time they have spent in the tissue, the cumulative (micro)environmental signals they are exposed to during their lifespan, and any (epi)genetic changes resulting from these exposures. We believe that studying mast cells within this framework will help resolve persisting controversies and unravel the many aspects of their biology that remain poorly understood.
| What is the relative contribution of different progenitors to mast cell (sub)populations? While it is now firmly established that the first mast cells originate from EMPs produced in the extra-embryonic YS, it is less clear to what extent these and subsequent progenitor waves contribute to the various mast cell compartments throughout life. Existing data from several fate mapping systems are best reconciled in a model in which most mast cells found in adult connective tissues originate from fetal-restricted progenitors, with a sizeable contribution of YS EMPs. The remainder of connective as well as a proportion of mucosal tissue-resident mast cells originates from either fetalrestricted or adult-type HSCs, or a mixture of both. To distinguish these possibilities, additional fate-mapping models are required that better differentiate these successive hematopoietic waves. Future studies on their origins will also need to better appreciate mast cell (sub)populations, such as those in the lung marked by presence or absence of integrin α4β7. Progenitors with mast cell potential are also present in mouse and human BM, but their contribution is tissue-and potentially environment-specific. At steady state, most mast cell populations in mouse connective tissue are only minimally supplemented from the BM. While mucosal sites recruit more BM progenitors, the overall contribution during adult homeostasis remains considerably lower than for many other tissue-resident lineages. At this point, we do not know exactly which signals drive BM recruitment at homeostasis. Considering that the abundance of BM-derived mast cells is highest in the gut and lung mucosa, the microbiome may be a factor. On the other hand, skin-resident mast cells are largely self-maintained with neglectable BM input. It is therefore important to comprehensively (re)-assess the contribution of BM progenitors to mast cells across the lifespan and sites, again taking into account subpopulations that may exist even within the same organ. Beyond the steady state, we should determine to what extent mast cells are generated de novo from the BM in response to infection or acute inflammation, whether BM-derived mast cells persist following resolution of these insults and how they compare to pre-existing mast cells. Similar considerations apply to chronic inflammatory conditions and cancer. Finally, it will be invaluable to expand on pioneering (single cell) transcriptomics studies in humans, using, for example, deep sequencing for somatic mutations in mitochondrial or nuclear DNA, technologies that provide a readout for cellular origins and lineage relationships.
| When, where, and how are mast cells specified from fetal progenitors? Despite continued progress in deciphering mast cell development, we still do not fully understand when, where, and how fetal (-derived) mast cells are specified from their progenitors. This is in part because the kinetics with which mast cells and their progenitors colonize fetal tissues are incompletely resolved. Mast cells can first be detected in humans from the 8th gestational week onwards. Relative to the length of gestation, this is earlier than in rodents, where the first granule-containing mast cells have been identified at E12.5, more than halfway through fetal development. In both humans and rodents, mast cells also seem to colonize different sites with distinct kinetics. These kinetics could potentially reflecting stage-specific demands for mast cells. To better understand these, we thus need to comprehensively map when mast cells appear across fetal tissues and developmental stages using a combination of transcriptomics, immune phenotyping, and imaging approaches. Mast cell progenitors appear in peripheral tissues before mature mast cells, indicating that fetal progenitors commit to the mast cell lineage prior to entering tissues and undergo further maturation there, much like what has been shown for adult progenitors. Although the core transcriptomic program that defines mast cells has been identified, 15 we however do not know which signals instruct progenitors to initiate this lineage-specific program. Considering their heterogeneity, it is likely that tissue-or population-specific signatures are superimposed onto this core mast cell program during in situ maturation. Future work will need to identify the underlying molecular mechanisms, the tissue-specific cues driving this, and the ways in which these are sensed and integrated by mast cells. We believe much can be learned from the comparison with macrophage specification, work on BM progenitors as well as the growing body of literature on human mast cell development. Finally, it is also still uncertain if there is a direct route for mast cell progenitors from the YS to peripheral tissues, like for some macrophages, or if instead, a fetal liver step is always required. There is evidence for both scenarios, and it is possible therefore that mast cell progenitors first follow a direct route from the YS and later are recruited from the fetal liver. However, this remains to be experimentally demonstrated.
| Do mast cells have developmental functions? Are these co-opted in pathology? Their "layered" ontogeny suggests that mast cells may be recruited from available hematopoietic progenitors to meet the demands of F I G U R E 3 Functional implications of mast cell ontogeny for health and disease. (Centre) Mast cells may be generated from available progenitors to meet stage-specific tissue demands. During normal development, they may support tissue remodeling processes. These physiological functions may be pathologically co-opted by exposure to adverse stimuli during development, or for example by the tumor microenvironment. In disease, additional mast cells may be generated from the bone marrow and aberrantly imprinted. (Top) Mast cells support developmental processes taking place before, around, and after birth. In the developing cornea, they mediate fine-tuning of vascular and neuronal networks. In the pre-optic region of the brain of male mice, mast cells responding to hormones mediate synaptic patterning. During puberty and pregnancy, mast cells also support branching morphogenesis in the mammary gland and spiral artery remodeling in the uterus. (Bottom) Fetal mast cells are primed in utero by maternal allergy. This renders them hyperactivated, resulting in heightened offspring susceptibility to allergic disease. Mast cells with different developmental origins and microenvironmental imprinting could mediate their proand anti-tumorigenic effects in cancer. Further explanations in the main text. specific developmental stages. It is thus conceivable that mast cells may have different functions throughout life. Before the adaptive immune system is established, one such demand is to protect the developing individual from pathogens and other immunological challenges, such as venoms. At least in the wild, these still pose a frequent threat to mammals. If mast cells were important for protective immunity in the pre-and perinatal period, this could be a reason why they are already present in the fetus, and why these cells have been maintained during evolution. In our view, an even more compelling reason would be if mast cells were involved in processes intrinsic to development. Although they are currently thought to be dispensable for normal development, there is evidence that they do contribute to tissue remodeling processes in the fetal cornea and pubertal mammary gland. It remains to be confirmed if these processes are truly mast cell-dependent, and whether mast cells more broadly support tissue maturation and remodeling. However, in keeping with this idea, data obtained in Kit-independent mouse models also found a role for mast cells in artery remodeling in the pregnant uterus. Whatever roles mast cells may play in development and normal tissue functioning, it seems possible that these can be co-opted in pathological conditions. In turn, their pathological actions, for example, in allergy, atopic disease, and cancer may "echo" their physiological functions. For example, sensitization of fetal mast cells by maternal IgE results in an increased allergen sensitivity postnatally. This exaggerated responsiveness represents a case of developmental programming but may also reflect a beneficial role for mast cells during normal development, which could prepare for rapid responses to harmful stimuli after birth, provided that fetal(-derived) mast cells are appropriately primed in utero. Finally, it is possible that mast cells established from different sources at distinct life stages may retain functional differences into adulthood. These may be cell-intrinsic, due to distinct environments experienced over their lifetime, or a combination of both. Such differences may be subtle or not at all apparent at steady state, however, they may become functionally relevant in non-homeostatic conditions such as wound healing or cancer. 1600065x, 2023, 1 , 33 CHIA 133 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | et al.
These were phenotypically identified by high expression of Kit, low-level expression of Thy1 and a low density of granules. They also express transcripts for three different mast cell proteases, Cpa3, Mcpt2, and Mcpt4. These Kit high Thy1 low cells are first detected at E14.5, peak in numbers at E15.5 and decline towards birth. In vitro, they efficiently produce mast cell colonies, but not any other lineages. In vivo, they repopulate peritoneal mast cells upon intra-peritoneal injection into deficient W/Wv recipients. Based on these features, they were considered circulating fetal mast cell progenitors or "pre-mast cells" that have F I G U R E 1 Mast cell development in the mouse. (Top) Different progenitors generating mast cells. The first mast cell progenitors arise from the erythro-myeloid progenitors (EMPs) produced in the extra-embryonic yolk sac starting at embryonic day 8.25 (E8.25
1600065x, 2023, 1 , 35 CHIA 135 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | et al.fetal-restricted progenitors) thus remains to be fully elucidated.Moreover, it is unclear if integrin α4β7 is functionally required for tissue homing of these progenitors.
1600065x, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
replenishment, or expansion of tissue-resident mast cell populations can be achieved via several, complementary mechanisms. Progenitors can be mobilized from the BM or alternative sources, and/or recruited progenitors and (remaining) mature mast cells can (further) expand in the affected tissue by proliferation. The exact sources and cellular mechanisms underlying mast cell replenishment and hyperplasia in non-homeostatic conditions likely depend on the nature of the insult. In summary, mast cells are versatile immune cells that arise during fetal development and show considerable heterogeneity between and within tissues and in response to environmental perturbations. They are also heterogeneous with respect to their ontogeny. Connective tissue-resident mast cells originate from YS EMPs and likely later fetal-restricted progenitors. Once established, they self-maintain independently from the BM. Mucosal mast cells, on the other hand, are partially replenished from the BM over time. Mast cell phenotypes are thus likely shaped by their origins and the microenvironments they encounter. As we will discuss in the following section, their functions may be adapted to the challenges of distinct life stages, and can potentially be co-opted in pathological settings.
| 39 CHIA 39 et al.
1600065x, 2023, 1 , 1 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1600065x, 2023, 1 , 41 CHIA 141 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | et al.
1600065x, 2023, 1 , 1 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1600065x, 2023, 1 , 43 CHIA 143 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | et al.
1600065x, 2023, 1 , 1 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License carcinoma, fetal-derived macrophages characterized by expression of folate receptor beta (FOLR2) promote vascularization, proliferation, and an immuno-suppressive environment through selective interaction with endothelial cells expressing Plasmalemma Vesicle
1600065x, 2023, 1 , 45 CHIA 145 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | et al.
1600065x, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imr.13191 by Cochrane France, Wiley Online Library on [06/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
10.1155/2020/4293071
|
cc-by
| null | null |
openalex
|
Aim. This study was aimed at investigating the effects and molecular mechanisms of physical activity intervention on Parkinson's disease (PD) and providing theoretical guidance for the prevention and treatment of PD. Methods. Four electronic databases up to December 2019 were searched (PubMed, Springer, Elsevier, and Wiley database), 176 articles were selected. Literature data were analyzed by the logic analysis method. Results. (1) Risk factors of PD include dairy products, pesticides, traumatic brain injury, and obesity. Protective factors include alcohol, tobacco, coffee, black tea, and physical activity. (2) Physical activity can reduce the risk and improve symptoms of PD and the beneficial forms of physical activity, including running, dancing, traditional Chinese martial arts, yoga, and weight training. (3) Different forms of physical activity alleviate the symptoms of PD through different mechanisms, including reducing the accumulation of α-syn protein, inflammation, and oxidative stress, while enhancing BDNF activity, nerve regeneration, and mitochondrial function. Conclusion. Physical activity has a positive impact on the prevention and treatment of PD. Illustrating the molecular mechanism of physical activity-induced protective effect on PD is an urgent need for improving the efficacy of PD therapy regimens in the future.Introduction Parkinson's disease (PD) is a second common neurodegenerative disease all over the world . Most of the patients are between 50 and 60 years old. As the aging of population increases, the risk of Parkinson's disease increases accordingly, and the incidence in young-and middle-aged people increases. As a chronic disease, PD has a long course and is prone to recurrence. The reduction of dopamine (DA) is the leading cause of PD in previous studies . The consequent loss of the neurotransmitter DA in the striatum leads to the primary motor symptoms of PD, namely, bradykinesia, tremor, rigidity, and postural instability . At present, the clinical trials are valid only for symptom management; no medications have proved effective in stopping the disease process . Therefore, revealing the patho-logical mechanism of PD is extremely important for the effective prevention and treatment of this disease. The character of brain tissue from PD patients is the degeneration of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, with the concomitant loss of their axons which project to the striatum along the nigrostriatal pathway. Along with the neuronal loss, the appearance of insoluble cytoplasmic inclusions (Lewy bodies, LB) and insoluble fibrils (Lewy neurites, LN) is also a neuropathological hallmark of PD. The main composition of the LB and LN is the α-synuclein (α-syn) protein . PD can usually be divided into familial and sporadic . Clinical statistics show that the number of familial patients accounts for about 10% of all patients. The leading cause of familial patients is gene mutation, such as PARKIN, DJ-1, PINK1, and ATP13A2 , while the main causes of sporadic patients are associated with oxidative stress, neuroinflammation, mitochondrial dysfunction, and environmental factors, such as drugs and pesticides .
Epidemiology Study of Parkinson's Disease 2.1. Incidence of Parkinson's Disease. A study of data from 2005 to 2018 indicated that PD is one of the most common neurodegenerative diseases worldwide (second only to Alzheimer's disease). The influence factors in the study of PD were mainly divided into prospective studies and casecontrol studies. In the world's high-income countries, the median incidence of PD is 14/100,000, and the rate is 160/100,000 in people aged 65 or older . Among the 40-year-old American population, the risk of PD in men is about 2%, while in women is 1.3% . The age-adjusted prevalence of PD reflects morbidity and mortality, which in Africa is lower than that in Europe, the United States, and Asia . Currently, there are fewer morbidity data related to racial or ethnic, but the incidence varies according to the current research. A study from a large medical institution in the United States indicated that the incidence in Blacks is higher than that in Whites. The age-adjusted and gender-adjusted incidence of PD was highest among Hispanics (16.6/100,000), followed by non-Hispanic whites (13.6/100,000), Asian (11.3/100,000), and Blacks (10.2/100,000). Another study based on beneficiaries of US health insurance suggested that the incidence of PD in Whites was also higher than that in Blacks or Asians . The incidence of PD increases with age and reaches a maximum at 80 years old. In the Chinese population aged 60 years, the incidence of PD is more than 1%.
Risk Factors and Protective Factors of Parkinson's Disease. The risk factors of pathogenicity and morbidity in PD are various. We have found that dairy consumption is positively correlated with the incidence of PD through a series of studies related to aging, cancer prevention, and nutrition . The risk of PD in the study sample from the Honolulu-Asian Ageing Study (HAAS) and Cancer Prevention and II Nutritional Research (CPS-IIN) increased with the extension of plantation time [21, 22] . This result is consistent with agricultural health research, which indicates that exposure to pesticides increases the risk of PD. Pesticides could cause oxidative stress and disturb mitochondrial function . Traumatic brain injury can lead to disruption of the blood-brain barrier, impaired mitochondrial function, and accumulation of brain α-syn protein, all of these may lead to an increased risk of PD after exposure to such injury . A cohort study in Finland found that overweight (i.e., BMI 27-29.9) or obesity (i.e., BMI >= 30) will bring a high-risk factor for PD [25, 26] . The protective factors of PD from numerous studies are also diverse. Compared with nondrinkers, longitudinal studies support a viewpoint that drinkers had a slight decreased risk of PD, which was consistent with the effect of alcoholic beverages on urate levels in the body [27, 28] . Coffee drinkers have a lower risk of PD than other people who do not drink coffee, which has been confirmed in several prospective investigations [29, 30] . The ingredients of black tea may help reduce the risk of PD, but green tea does not have the same effect . With the extension of smoking time, the risk of PD is reduced by up to 70%, and it has increased with time since the quitters quit smoking . Besides, the use of antihypertensive drugs [36, 37] , physical activity, and a healthy diet can effectively reduce the risk and relieve the symptoms of PD patients (Figure 1 ).
Physical Activity Prevention and Parkinson's Disease . There is no strong supporting evidence for the hypothesis that physical activity can prevent male PD in the Harvard Alumni Health Study. Nevertheless, a smaller sample size study shows a negative and nonsignificant association between physical activity and PD . A study of 143,325 participants from CPS-II-N has found that vigorous activity was associated with PD in men and women, while a reduction in PD risk through moderate to vigorous activity . The study of 213,701 participants of NIH-AARP Diet and Health Study cohort also confirmed this view that higher levels of moderate to vigorous activities at ages 35-39 or in the past ten years as reported in 1996-1997 were associated with low PD incidence after 2000, which was with a significant dose-response relationship. Compared to individuals who were inactive during the two periods, the risk of PD reduced by approximately 40% in the further analysis . Another study from the Swedish National March cohort showed that the total amount of daily activity was associated with a lower risk of PD, but women's correlation was not apparent to men . . Physical activity treatment could also improve nonmotor functions in PD patients. In the meantime, physical activity could regulate autonomic dysfunction, including improvement of cardiac sympathetic regulation in PD patients , but the effects on other systems are unclear . Resistance training, Qigong, and other types of physical activity could improve sleep quality in PD patients . Another benefit of physical activity to PD patients is cognitive impairment alleviation. David et al. found that 24 months of progressive resistance training may improve attention and working memory in nondemented patients with mild to moderate PD patients . McKee and Hackney indicated that tango participants improved on disease severity, spatial cognition, balance, and executive function . Consistent with the above studies, Cruise et al. found that 12 weeks of progressive aerobic and anabolic physical activity had selective benefits for cognitive functioning by improving frontal lobe-based executive function . As a safe, broadspectrum intervention, physical activity could also have positive effects on mood, cognition, and sleep for PD patients. Therefore, physical activity could enhance the chances of recovery through improvement in the mood and the nervous system in elderly PD patients . Based on the Feldenkrais method, Teixeira-Machado et al. indicated that 50 sessions of physical therapy programs could promote cognitive function for ages between 50 and 70 PD patients . More and more evidences from numerous studies suggested that physical activity could enhance motor and nonmotor symptoms of PD patients, but the mechanism of exercise in relieving PD symptoms needs further study.
Multiple Physical
Therapy Regimen by Comparing the Effects of Different Intensities of Physical Activity on PD. The light physical activity (VO 2Max 40%-50%) reduces tumor necrosis factor alpha (TNF-alpha) in the skeletal muscle and the thiobarbituric acid reactive substances (TBARS) in the soleus muscles . Linke's study found that physical activity can not only reduce the expression of inflammatory cytokines in the blood but also enhance the activity of free radical scavengers . Schulze's findings suggest that physical activity enhances mitochondrial biogenesis in the vascular endothelium through a shear stress-dependent mechanism . Vettor's findings demonstrate that physical activity promotes endothelial NO synthase-(eNOS-) dependent mitochondrial biogenesis in the heart, which behaves as an essential step in cardiac glucose transport . The light activity can reduce oxidative stress and enhance mitochondrial biogenesis in skeletal muscle, blood, and heart. These protective effects can improve autonomic dysfunction , sleep quality , and depression in PD patients. The moderate to vigorous physical activity (VO 2Max 50%-80%), 40-60 mins/day, 5 days/week, can be widely used in people's daily life, corresponding to about 7 hours of walking, 5 hours of aerobics, or 3 hours of lap swimming per week for men and 6 hours of walking, 4.5 hours of aerobics, or 2.5 hours of lap swimming per week for women [44, 45] 3 Oxidative Medicine and Cellular Longevity physical activity (exercise) has a more effective protection mechanism in PD patients. The moderate treadmill exercise and one-time exhaustion exercise, in addition to effectively reducing the risk of PD , can also enhance motor deficits in PD patients , improve cognitive impairment, and depression , which is consistent with the role of light physical activity. Besides, animal experiments demonstrated that moderate to vigorous physical exercise could enhance mitochondrial function and reduce oxidative stress [70, 71] in the brain of PD mice. More importantly, exercise can reduce the accumulation of the pathogenic protein α-Syn and prevent neuronal apoptosis .
Molecular Mechanisms of Physical Exercise Relieving Parkinson's Disease 4.1. Physical Exercise Can Reduce the Accumulation of the α-Syn Protein. The α-syn protein is the main pathogenic protein of PD, which is acidic synaptophysin expressed in the vertebrate presynaptic. In the central nervous system (CNS), many neurodegenerative diseases are associated with the exiting of the α-syn protein in the cytoplasm and nucleus . Aggregation of α-syn is a crucial risk factor for PD, multiple system atrophy (MSA), and Lewy body dementia (DLB) [73, 74] . In previous studies, physical exercise was found to have a positive effect on neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and PD . Physical exercise could reduce the loss of dopaminergic neurons, increase synaptic connections, and upregulate neurotrophic factor levels to improve PD dyskinesia . Physical exercise could downregulate α-Syn protein levels and neuronal apoptosis [70, , which could reduce inflammation and mitochondrial dysfunction to restore the motor function in PD patients. Overexpression of α-synuclein also resulted in significant impairment on hippocampal neurogenesis-dependent pattern separation (a cognitive task). Voluntary running exercise could prevent deterioration and improve cognition through the decrease of α-Syn protein overexpression. This can be further substantiated by an effect of running on neurogenesis levels in the dorsal dentate gyrus, suggesting that the functional effects of running on pattern separation were mediated via increased neurogenesis . However, the exact molecular mechanisms of exercise-induced α-syn protein level decrease are unrevealed. The accumulation of the α-Syn protein is the main reason for neuron loss [85, 86] . Physical exercise can significantly reduce α-Syn protein neuron loss in PD rodent models [70, 82, 83] , but there are still inconsistent results from other studies [87, 88] . The different extent to the loss of neurons may be due to changes in exercise duration and intensity, which could affect the motor benefits of PD patients . Aguiar et al. found that six weeks of running did not prevent MPTP neurotoxicity, suggesting that the duration of physical exercise should be prolonged to induce a neuroprotection effect . Therefore, shortterm, low-intensity physical exercise is not sufficient to alleviate neuronal loss , but moderate to intense intensity of physical exercise may have the protective effect on neurons from loss [70, 91, 92] . Active physical exercise can alleviate neuron loss and enhance nerve regeneration. In the substantia nigra pars compacta and striatum brain regions, exercise increases the levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) [82, , which will promote the expression of PSD-95 and synaptophysin [78, 97, 98] . These positive effects also could increase the function of dendritic spines on dopaminergic neurons and nerve fibers . Current studies have found a significant reduction in neurogenesis in the hippocampus of PD patient, while a significant increase in neurogenesis after a period of running. Increased neurogenesis in the hippocampus promoted learning ability and memory function . Both acute and chronic physical exercises could increase hippocampus activity [106, 107] . Exercise improves hippocampal synaptic plasticity mainly due to enhanced synaptic efficacy and expression of molecules involved in learning and memory . Physical exercise not only promotes cell proliferation but also promotes the differentiation of newly formed nerve cells. Physical exercise increases neurogenesis, enhances synaptic plasticity in neurons, and improves spatial memory.
Physical Exercise Can Reduce Inflammation and Oxidative Stress. PD is a very common neurodegenerative disease in the elderly that is characterized by skeletal muscle abnormalities [111, 112] . Related studies reported that the inflammatory factors were upregulated in PD brain . In addition, overexpression of inflammatory factors has also been shown in gastrocnemius skeletal muscle in PD, which suggested that they may play a role in the progression of skeletal muscle abnormalities . Physical exercise training can reduce the risk of inflammation . Erekat et al. and Al-Jarrah et al. found PD-induced changes in skeletal muscle IL-1β and TNF-α inflammatory cytokine expression. This is consistent with previous studies that skeletal muscle fibers are capable of producing proinflammatory factors [118, 127] . These proinflammatory cytokines are overexpressed in PD-induced skeletal muscle cells [116, 128, 129] . However, after physical training, overexpressed inflammatory cytokines are inhibited in aging skeletal muscle [64, 122] . Previous reports suggest that physical training promotes the expression of antioxidant enzymes, which may help reduce the production of proinflammatory cytokines in the skeletal muscle. Regular physical exercise can upregulate cellular antioxidant capacity and reduce the production rate of reactive oxygen species (ROS) 130] . Besides, physical exercise can increase the number and function of mitochondria to induce mitochondrial biogenesis [68, , thereby improving the oxidative environment caused by mitochondrial abnormalities, which are associated with inflammatory reactions occurring in PD skeletal muscle . Therefore, it can be assumed that mitochondrial biogenesis induced after physical exercise training leads to a decrease in proinflammatory factors in the skeletal muscle in PD patient [137, 138] . Oxidative Medicine and Cellular Longevity Accumulation of the α-syn protein in neurons leads to inflammation in the brain . Aggregation of α-Syn induced the production of proinflammatory cytokines, which are toxic and thus cause cell death of dopaminergic neurons [142, 143] . Therefore, reducing the inflammatory response may be an effective way to deal with PD. Several studies found that physical exercise can improve the oxidative metabolism and the expression of antioxidant enzymes in the brain of mice . Tuon et al. indicated that physical exercise was beneficial in reducing the production of proinflammatory proteins and inflammation in the brain of PD mice . This result is consistent with previous studies by Sung et al. and Al-Jarrah et al., which suggested that physical exercise could decrease the level of proinflammatory proteins in the striatum and hippocampus in the PD experimental model through reducing the activity of microglia [147, 148] . Based on the above studies, we speculate that multiple pathways are involved in the regulation of physical exercise in PD animal experiments, such as alleviate the production of proinflammatory factors in the musculoskeletal muscle, reduce the expression of inflammatory factors in the brain, reduce the inflammatory response, and regulate oxidative stress [149, 150] .
Physical Exercise Can Increase the Upregulation of Brain-Derived Neurotrophic Factor (BDNF). The neuroprotective effects of exercise in PD may be promoted by neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). BDNF plays a vital role in cell differentiation, neuronal survival, migration, synapse development, and synaptic plasticity . Studies have shown that neurogenesis, combined with BDNF, can mimic the beneficial effects of physical exercise on mice . Many studies have shown that physical exercise can produce antidepressant effects by increasing the level of BDNF, as well as prevent neurodegenerative diseases . Running has been shown to increase BDNF and nerve growth factor (NGF) , fibroblast growth factor-2 (FGF-2) , and insulin-like growth factor 1 (IGF-1) . Striatum trophic factors have potent trophic activity on DA neurons . For example, 6-OHDA could prevent neurotoxic effects . Physical exercise-trained rats showed an increase in protein and mRNA levels of trophic factors in the brain [153, 163] , which suggested that this neuroprotection is caused by an increase in trophic factors [164, 165] . Besides, the protective effects induced by physical exercise are not limited to this aspect. We noted that FGF-2, a trophic factor, is upregulated after physical exercise and has also been shown to induce expression of GDNF and BDNF in vitro [166, 167] . Physical exercise could increase the levels of BDNF and other neurotrophic factors, while toxins in the striatum and hippocampus could decrease those factors [168, 169] . In summary, studies have shown that physical exercise leads to the upregulation of neurotrophic factors in the brain of PD mice and enhances neuronal survival, differentiation, and synaptic plasticity in the central nervous system.
Physical Exercise Can Enhance Mitochondrial Function. Mitochondria are essential dynamic organelles responsible for the production of a large number of cellular energy molecules, adenosine triphosphate (ATP). Mitochondria also often undergo fission, fusion, and biogenesis and maintain tubular networks under normal conditions. These dynamic processes play an essential role in neuronal survival and homeostasis . The common hallmark of several neurodegenerative diseases (Huntington's disease, Alzheimer's disease, and PD) is impaired function or expression of PGC-1α, a major regulator of mitochondrial biogenesis . Recent studies have found that disrupting mitochondrial dynamics (e.g., excessive fission and repressed biosynthesis) leads to mitochondrial dysfunction in PD, which triggers neuronal cell death . More and more researches have begun to reveal that endurance aerobic exercise can improve mitochondrial function in the brain . Cells exposed to neurotoxins showed mitochondrial rupture, reduced mitochondrial protein, and cell death. Aerobic exercise could change mitochondrial phenotype, such as upregulating antiapoptotic protein (MCL-1 and BLC-2) and reducing proapoptotic proteins [70, 176] . Also, physical exercise could regulate fusion (such as MFN1/2 and OPA1) and fission and enhance mitochondrial biosynthesis to promote mitochondrial dynamics . The number of autophagic vacuoles increased in neurons in the brain regions of PD patients. This result indicates that the autophagy of neurons in the brains of PD patients is higher than that of the healthy population . Koo et al. found that running exercise promotes autophagic clearance of mitochondrial α-Syn by activating mitochondrial silencing signal regulator-1 (SIRT1) . Jang et al. suggested that eight weeks of running exercise regulates levels of autophagy-associated proteins, including microtubule-associated protein 1 light chain 3-II, p62, BECLIN1, BNIP3, and lysosomalassociated membrane protein-2. Those factors were all downregulated in the PD mice group while reversed in the exercise group. Therefore, physical exercise relieve PD symptoms in multiple ways, such as the autophagic ability promotion of the cells and mitochondrial function enhancement.
Conclusion The process of PD is affected by various factors, including risk factors and protective factors. As a safe treatment, physical activity could relieve the symptoms in PD patients, such as motor dysfunction, cognitive deficits, and depression. Different forms of physical activity, especially the moderate to vigorous physical exercise, have a positive impact on PD through multiple mechanisms, including reducing the accumulation of the α-syn protein and alleviating inflammation and oxidative stress, while enhancing BDNF activity, nerve regeneration, and mitochondrial function. So, there is an urgent need for illustrating the molecular mechanism of physical activity-induced protective effect on PD in order to provide the theoretical basis for improving the efficacy of prevention and treatment of PD (see Figure 1 ). . Compared with light physical activity, moderate to vigorous Figure 1: A schematic of physical activity on Parkinson's disease. Risk factors for Parkinson's disease include dairy intake, prolonged exposure to pesticides, traumatic brain injury, and obesity. The protective factors on Parkinson's disease include alcohol intake, tobacco smoking, coffee, black tea, and physical activity. Common forms of physical activity that benefit Parkinson's disease include running, Obesity Black tea Traumatic Coffee Dairy products Risk factors Parkinson's disease Protective factors Physical activity Insecticide Smoking Pesticide Alcohol Autonomic dysfunction Sleep quality Depression Motor Cognitive impairment α-Syn and oxidative stress Mitochondrial function aggregation and neuronal loss BDNF expression Autophagic cell death The light physical activity The moderate to vigorous physical exercise (the protective effect is mainly (the protective effect is not only in the muscle, in muscle, blood and heart) blood and heart but also in the brain) dancing, traditional Chinese martial arts, yoga, and weight training. Physical exercise can reduce α-Syn protein aggregation, alleviate neuronal death, regulate inflammation and oxidative stress, promote BDNF activity, modulate neuronal autophagy, and enhance mitochondrial function. Physical activity can improve motor capacity including strength, balance, and flexibility and also enhance the nonmotor symptoms, alleviate cognitive impairment, and improve depression.
|
10.1371/journal.pone.0161507
|
cc-by
| null | null |
openalex
|
Immunomodulatory Foxp3 + regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2 + Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2 + Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 -especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2 + Tregs are superior to ST2 -Tregs in suppressing CD4 + T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2 + Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2 + Tregs useful targets for immunomodulatory therapies.Introduction Regulatory Foxp3 + CD4 + T cells (Tregs) are key controllers of immune homeostasis. They maintain immune tolerance, thus preventing autoimmunity or excessive inflammation [1, 2] . They are present in almost all tissues, even under homeostatic conditions, and regulate a variety of innate and adaptive immune cells [3, 4] . Various mechanisms mediating Treg functions have been described. These include direct suppression or cytolysis of target cells, repression of APC maturation and function as well as secretion and activation of anti-inflammatory cytokines such as IL-10 and TGFβ [5, 6] . Consequently, Tregs form a heterogeneous population displaying diverse migratory properties and immunomodulatory effects. A minor fraction of Tregs in the circulation and lymphatic organs exhibits an activated effector/memory T cell phenotype similar to conventional T cells, thus termed effector Tregs. These Tregs are assumed to have encountered antigen more recently and preferentially reside in non-lymphoid tissues (NLT) . Several surface markers distinguishing effector Tregs have been identified so far, including αE integrin (CD103) which marks a subset of highly suppressive, rapidly activated Tregs that preferentially resides in NLT . A similar phenotype is observed in KLRG1-expressing Tregs that accumulate in the lung in a model of airway inflammation, accompanied by increased levels of CD44, CD69, CD25, CTLA-4 and a downregulation of CD62L [11, 12] . In general, effector Tregs display classical T cell activation markers, like CD44 hi and CD62L lo , along with molecules involved in Treg maintenance and function, such as Foxp3, CTLA-4, KLRG1, CD103 and ICOS and are thought to be highly suppressive. A growing body of evidence suggests that the acquisition of an effector-like phenotype does not mark the end point of Treg differentiation. Instead, further diversification comparable to conventional T cells may occur . Notably, the Th2 lineage-specifying transcription factor GATA-3 can be upregulated in Tregs upon encounter of antigen and IL-2 [17, 18] . In Th2 cells, GATA-3 induces transcription of the Il1rl1 gene, encoding ST2, the receptor for the alarmin IL-33 . Recently, it was shown that ST2 is also expressed on a subset of Tregs in a GATA-3-dependent manner . Additional studies revealed that systemic administration of IL-33 increased the number of total and ST2 + Tregs resulting in a delay of graft-versus-host disease and amelioration of colitis . Moreover, in a setting of acute inflammation, IL-33 signals are essential for the accumulation of ST2 + Tregs in mucosal tissue and the stability of the Treg phenotype . Yet, ST2 + Tregs are present in several organs even under homeostatic conditions , but their phenotype and suppressive capacity at steady-state remain ill defined. Here we report that ST2 + Tregs are highly activated effector Tregs that preferentially accumulate in NLT. They exhibit a Th2-like phenotype with elevated expression of GATA-3 and production of the Th2 cytokines IL-5 and IL-13, which can be further augmented by IL-33 signals. In line with their effector-like phenotype, ST2 + Tregs suppress naïve CD4 + T cell proliferation more effectively than their ST2 -counterparts-independent of IL-33. Both IL-10 and increased TGFβ production and activation contribute to the suppressive mechanism employed by ST2 + Tregs. Finally, we demonstrate that IL-33 is dispensable for the generation, maintenance and tissue accumulation of ST2 + Tregs under homeostatic conditions. Taken together, ST2 + Tregs form a highly suppressive subset located in prime position to react to inflammatory processes involving the release of IL-33.
Material and Methods
Mice Il33 -/- , Il1rl1 -/- and WT mice were bred on C57BL/6 background under SPF conditions at the Charité animal facility, Berlin. B6.Foxp3 hCD2 reporter mice were crossed to Il10 gfp reporter under SPF conditions at animal facilities at the University of Oxford, UK. Experiments were performed at the Charité and DRFZ, Berlin, in accordance with national law for animal protection with permission from the Landesamt für Gesundheit und Soziales (Lageso); permission number T0058/08. Experiments at the University of Oxford were approved by the Clinical medicine AWERB committee at the University of Oxford. Animals were euthanized by cervical dislocation.
T cell isolation for ex vivo characterization Blood was drawn from animals before they were euthanized and perfused with PBS. Circulating lymphocytes were isolated by high density centrifugation using Histopaque (Sigma-Aldrich). Lungs were chopped, treated with 0.1U/ml Collagenase D (Roche), homogenized and applied to a Histopaque gradient. Spleen and LN were homogenized and splenic erythrocytes were lysed. Payer's patches were removed from the small intestine (SI). Colon and SI were cut and incubated in RPMI with 1mM DTT, 5mM EDTA, 5% FCS, homogenized and treated with 0.1U/ml Collagenase D. Single cell suspensions were applied to a 40/70% Percoll gradient.
Flow cytometric analysis and sorting Samples were stained with antibodies against CD4 (GK1.5), CD25 (PC61), KLRG1 (2F1), CD103 (2E7), CD62L (MEL-14), CD44 (IM7), LPAM-1/α4β7 (DATK32), CD183/CXCR3 (CXCR3-173), CD194/CCR4 (2G12), CD196/CCR6 (29-2L17), CD197/CCR7 (4B12), CD199/ CCR9 (CW-1.2), anti-hCD2 (RPA-2.10) and ST2 (DJ8) in PBS/0.2% BSA/2mM EDTA. ST2 staining was amplified using FASER-Kit-PE or FASER-Kit-APC (Miltenyi Biotec). CTLA-4 (UC10-4B9) staining was performed after fixation. T-bet (4B10), GATA-3 (TWAJ), and Foxp3 (FJK-16S) were stained using the Foxp3 staining buffer set (eBioscience). GATA-3 index depicts the MFI ratio of GATA-3 and its respective isotype (eB149/10H5) staining. Samples were acquired on FACSCanto II (BD Biosciences) and analyzed using FlowJo (FlowJo) or FCAP array software (BD).
T cell cultures and suppression assays CD4 + T cells from spleen and LN were enriched by magnetic cell separation (MACS, Miltenyi Biotec). CD4 + CD25 + ST2 + or CD4 + CD25 + ST2 -T cells (Tregs) and CD4 + CD62L + CD44 lo (Tresp) were purified by FACS. For Treg stimulation, 96-well flat-bottom plates were coated with 3μg/ml anti-CD3 (145-2C11) and 6μg/ml anti-CD28 (37.51) antibodies. 2x10 4 to 1x10 5 Tregs were resuspended in 140-200μl of RPMI/10% FCS supplemented with 40ng/ml IL-2 with or without 30ng/ml IL-33. After 60-70h, supernatants were collected and cytokines were quantified by cytometric bead array (BD Pharmingen). For suppression assays, Tresp were labeled with CellTrace Violet or CFSE (Invitrogen). APCs were isolated from splenocytes by MACS using biotinylated anti-CD19 (1D3), anti-CD11b (M1/70) and anti-CD11c (HL3) antibodies and anti-biotin microbeads (Miltenyi Biotec). IL-10 signaling was blocked by pre-incubation of Tresp with 50μg/ml anti-CD210/IL-10R (1B1.3a). TGFβ signaling was blocked using 10μM TGFβRI inhibitor in DMSO (SB431542, Merck Millipore) directly in culture. Control cells were treated with DMSO alone. The Treg: Tresp ratio was varied as indicated; twice as much APCs and 5μg/ml anti-CD3 antibody were added. Proliferation of Tresp was analyzed on day 4 by flow cytometry. The division index indicates the average number of divisions that a cell in the starting population has undergone. The relative division index was normalized to the corresponding untreated population.
mRNA isolation and quantification RNA was isolated using the miRNeasy mini kit (Qiagen). Complementary DNA was synthesized using the Taqman Reverse Transcription Reagents (Life Technologies). Quantitative RT-PCR reactions were performed in duplicates using SYBR Select Master Mix (Life Technologies), respective primers (S1 Table ) on a QuantStudio 7 real-time PCR system (Life Technologies). Data was normalized to Hprt endogenous control. All reagents and kits were used at manufacturer's recommendation, if not stated otherwise.
Statistical analysis Two groups were compared with two-tailed unpaired Student's t test (GraphPad Prism 5.02); Ã p 0.05; ÃÃ p 0.01; ÃÃÃ p 0.001; non-significant (ns) p > 0.05. First, CXCR3, involved in Treg recruitment to inflammatory sites , was significantly higher expressed on ST2 + than ST2 -Tregs in all organs analyzed but the lung, suggesting an increased potential of ST2 + Tregs to home to those organs. However, expression was highest in the lung hinting towards a general requirement for lung-homing Tregs to express CXCR3. Second, CCR9 and α4β7 integrin are strong promoters of lymphocytic migration to the intestinal lamina propria . Their expression was significantly higher on ST2 + than ST2 -Tregs found in the gut. Third, CCR4 has been implicated in enhanced migration of Tregs to the skin and lung to control local inflammation . We found significantly higher CCR4 expression on ST2 + Tregs, suggesting preferential migration to these sites. Last, CCR7, a homing marker for secondary lymphoid organs [32, 33] , was significantly lower expressed on ST2 + than ST2 -Tregs. Taken together, these results indicate that ST2 + Tregs accumulate in NLT, presumably facilitated by their distinct chemokine receptor and integrin expression pattern.
Results
ST2 + Tregs arise independently of IL-33 signals
ST2 + Tregs display a highly activated phenotype Next, we analyzed the expression of molecules associated with Treg function and activation on ST2 + and ST2 -Treg subsets. Both KLRG1 and CD103 have been described to be expressed on highly suppressive Tregs with the capacity to home to inflamed tissues . Around 70% of splenic ST2 + Tregs expressed either one or both of these markers compared to only about 10% in the ST2 -Treg compartment. A similar distribution was observed in the pLN and lung (Fig 2C ). These data suggest that the ST2 + Treg subset is largely comprised of highly differentiated and suppressive Tregs. To provide further evidence for the activated phenotype of ST2 + Tregs, two classical activation-associated surface molecules were assessed. While CD44 expression on Tregs is correlated with increased suppressive capacity , the role of CD62L in Treg functionality is controversially discussed . We found that ST2 + Tregs expressed high levels of CD44 and low levels of CD62L. Additionally, CTLA-4 is crucial for the suppressive function of Tregs . Indeed, the frequency of CTLA- At the mRNA level we could confirm a higher expression of Il1rl1 (ST2) and Foxp3 in ST2 + Tregs (Fig 2F ). Furthermore, the expression of two molecules associated with a highly activated and suppressive Treg phenotype, Icos and Prdm1 (Blimp1) [39, 40] , was significantly increased in the ST2 + compared to the ST2 -Treg subset. Next, we assessed the production of the antiinflammatory cytokine IL-10, whose expression in Tregs under homeostatic conditions is dependent on Blimp1 and has been shown to confer Treg function . In all organs, ST2 + Tregs displayed an increased capability to produce IL-10 when compared with ST2 - Tregs, with greatest differences detectable in the lung (Fig 2G ). Overall, these data suggest that the ST2 + Treg subset combines various characteristics of highly activated, differentiated and suppressive Tregs. proliferation at all ratios tested, ST2 + Tregs were considerably more suppressive (Fig 3A and 3B ). However, the addition of IL-33 had no beneficial effect on their suppressive capacity, but resulted in a selective increase of ST2 expression on ST2 + Tregs (Fig 3C ). Taken together, these results demonstrate that ST2 + Tregs strongly suppress CD4 + responder T cell proliferation. This property appears to be independent of their ability to receive and process IL-33 signals through the ST2 receptor.
ST2 + Tregs suppress responder T cells via IL-10 and TGFβ release and activation Both IL-10 and TGFβ are thought to at least partially mediate the suppressive function of Tregs . Since ST2 + Tregs produce significantly higher amounts of these cytokines than their ST2 - counterparts, we addressed if these cytokines contribute to the increased suppressive capacity of ST2 + Tregs. Therefore, we blocked IL-10 or TGFβ signaling during in vitro suppression assays (Fig 4E ). The blockade of IL-10 signaling by antibodies targeting the IL-10 receptor increased the proliferation of Tresp to a similar extent in the presence of ST2 + and ST2 -Tregs, hinting at similar contribution of IL-10 to the suppressive mechanisms of both Treg subsets despite differences in the secreted amount. In contrast, the application of a TGFβ receptor I inhibitor resulted in enhanced proliferation of Tresp cultured in the presence of ST2 + Tregs, whereas Tresp alone or cultured with ST2 -Tregs were only marginally affected. This finding suggests a major contribution of TGFβ signaling to the suppressive mechanism of ST2 + but not ST2 -Tregs. In fact, not only the production of total TGFβ was higher in ST2 + Tregs but also the expression of integrin αvβ8 (Itgav and Itgb8) associated with the activation of latent TGFβ (Fig 4F ). Taken together, these data demonstrate that ST2 + Tregs employ a mechanism based on IL-10 and increased TGFβ production and activation to at least partially mediate their highly suppressive function.
Discussion Recently, the expression of the IL-33 receptor ST2 was found on a subset of regulatory T cells [20-22, 24, 45] . However, their phenotype and tissue distribution under homeostatic conditions as well as the suppressive mechanism employed by these cells are still poorly understood. Here we report that ST2 + Tregs display a Th2-biased effector Treg phenotype and strongly suppress naïve CD4 + T cell proliferation; an effect partially mediated by the anti-inflammatory cytokines IL-10 and TGFβ. We observed that at steady-state, ST2 + Tregs preferentially accumulated at non-lymphoid tissues, such as the lung, the lamina propria of the small intestine and colon as well as the circulation. These findings are in agreement with previous data obtained in inflammatory settings that show an accumulation of ST2 + Tregs in the colon , the lung and especially the visceral adipose tissue (VAT) [24, 46] . IL-33 is constitutively expressed in the nucleus of epithelial and endothelial cells and released upon necrotic cell death [47, 48] . However, under homeostatic conditions IL-33 release is very limited. Thus, our data demonstrate that signaling via the ST2 receptor is dispensable for the generation, maintenance and tissue accumulation of ST2 + Tregs. Notably, VAT Tregs form an exception. This specialized, self-contained Treg subset, which also expresses the transcription factor PPARγ associated with adipocyte differentiation , is highly dependent on the IL-33/ST2 axis for their stability and function in the VAT [24, 46, 50] . Regarding ST2 + Treg tissue migration, we identified chemokine receptors and integrins associated with NLT homing to be particularly highly expressed in the ST2 + Treg compartment, including CXCR3, CCR4, CCR6, CCR9, αE (CD103) and α4β7 integrin. Migration along the respective chemokine gradients could contribute to the positioning and retention of ST2 + Tregs at mucosal surfaces already under homeostatic conditions, leaving them in prime position to react to IL-33 danger signals. Low-level release of IL-33 due to constant minor cell damage may contribute to the augmented ST2 expression per cell observed at barrier tissues. We and others [20, 22, 24] demonstrated that ST2 + Tregs belong to a subset of highly activated effector Tregs based on their accumulation at NLT and expression of typical T cell activation markers and molecules involved in Treg maintenance and function. Recently, a subset of effector Tregs expressing the B cell-associated transcription factor Blimp1 was characterized [24, 40] . The authors report that Blimp1 is essential for the production of IL-10 by effector Tregs. Indeed, we found high expression of Prdm1 (Blimp1) in the ST2 + Treg compartment, paired with an increased IL-10 production capability in vitro and in vivo. Moreover, transcriptome analysis of Blimp1 + and Blimp1 -Tregs revealed a strong enrichment for ST2 in the Blimp1 + Treg subset . Overall, these findings suggest a considerable overlap between the ST2 + and Blimp1 + effector Treg populations. The Th2 lineage-defining transcription factor GATA-3 is a known inducer of ST2 expression in Tregs . In line with previous reports , we detected particularly high levels of GATA-3 in the ST2 + Treg compartment. Furthermore, these cells also secreted increased amounts of Th2-associated cytokines IL-5 and IL-13. In contrast to IL-4 which is produced neither by ST2 + nor ST2 -Tregs, GATA-3 has been reported to directly bind to the proximal promoters of IL-5 and IL-13 resulting in their expression . Additionally the expression of these cytokines can be further augmented by IL-33. However, we did not detect significant changes in GATA-3 expression upon IL-33 signals implying a mere supportive role of ST2 signaling on GATA-3 function. In contrast, in vivo administration of IL-33 not only expands Tregs but also increases GATA-3 expression in the ST2 + Treg compartment , but such a setting is more complex and secondary effects may contribute to the GATA-3 upregulation. Although IL-33 signals significantly expanded or promoted the survival of ST2 + Tregs in vitro, the increased amounts of the Th2 cytokines IL-5 and IL-13 released by these cells were not only a result of this expansion, but also of increased cytokine transcription. First reports indicate that Th2 cytokine production by human Tregs results in an anti-inflammatory phenotype of alternatively activated macrophages . However, more studies are necessary to determine the full functional consequences of Th2 cytokine secretion by Tregs. In accordance with their effector-like phenotype [8, 9, 11] , ST2 + Tregs were superior in suppressing naïve CD4 + T cell proliferation compared to their ST2 -counterparts. Yet, the comparison of ST2 + and ST2 -Tregs expanded in vivo by IL-33 injection has revealed only insignificant differences in suppressive capacity between the groups [21, 22] . Severe alterations in the hematopoietic compartment upon IL-33 administration including a potential upregulation of ST2 on expanding non-effector Tregs may be the underlying cause of this discrepancy. In line with previous data , we observed a similar suppressive capacity of ST2-deficient and WT Tregs, either due to the low abundance of ST2 + effector Tregs within the WT Treg compartment or a similar effector Treg frequency in Il1rl1 -/-mice. Furthermore, the addition of IL-33 had no effect on the suppressive capacities of ST2 + and ST2 -Tregs, despite an expansion of ST2 + Tregs. Such suppressive inertia to IL-33 signals might be due to Tregs exerting their most suppressive effects immediately after stimulation, whereas the cell number only gradually increases. Thus, ST2 signaling seems dispensable for the high suppressive function of ST2 + Tregs derived from secondary lymphoid organs. However, in NLT-derived ST2 + Tregs which express ST2 at a higher per cell amount, IL-33 signaling might be able to enhance their suppressive capacity. Notably, IL-33 has recently been associated with Treg-mediated wound healing in a number of different tissues [56, 57] . Injury-induced release of IL-33 from the parenchyma resulted in the release of tissue-protective epidermal growth factor ligand amphiregulin from ST2 + Tregs . Thus, ST2 + Tregs might present dual functionality towards limiting local tissue inflammation: on the one hand by strongly suppressing pro-inflammatory T cell responses and on the other hand by directly triggering tissue repair processes in an IL-33-dependent manner. A variety of mechanisms have been proposed to mediate Treg suppressive function: (1) direct target killing or suppression via IL-2 deprivation, granzyme B secretion and CD39/ 73-mediated ATP reduction; (2) repression of APC maturation and function via CTLA-4 and Lag3 signaling; and (3) secretion and activation of anti-inflammatory cytokines IL-10, TGFβ and IL-35 [5, 6] . Indeed, we detected high expression of CTLA-4, IL-10 and TGFβ in the ST2 + Treg subset, while others have already identified high levels of Lag3 and CD39 . Furthermore, we provide evidence that both IL-10 and to a greater extend TGFβ contributed to the suppressive capacity of ST2 + Tregs. High TGFβ and integrin αvβ8 expression was detected in ST2 + Tregs indicating an advantage of ST2 + Tregs to produce and activate latent TGFβ . In agreement with the unresponsiveness of ST2 + Tregs to IL-33 concerning their suppressive capacity, IL-10 and TGFβ secretion also remained unaffected. Additionally, absence of APCs from the suppression assay system did not alter the suppressive capacity of ST2 + Tregs (data not shown) suggesting that under these conditions, ST2 + Tregs directly suppress their target cells. However, the role of IL-10 and TGFβ in suppressing Tresp proliferation in vitro remains controversially discussed, with some reports claiming a contribution of these cytokines to the suppressive mechanism of Tregs [58, 59] and others not . Yet, the amount of IL-10 and TGFβ produced by the studied Treg populations might strongly influence the observed effect of these cytokines on suppression. As already mentioned, ST2 + effector Tregs produce higher amounts of IL-10 and TGFβ compared with their ST2 -counterparts. Thus, blockade of IL-10 and TGFβ signaling can exert a greater effect on ST2 + Treg-mediated suppression, which is readily detected in in vitro suppression assays. However, the reduction in suppression upon IL-10R blockade was comparable between ST2 + and ST2 -Tregs, despite greater IL-10 production by ST2 + Tregs. This observation might be explained by an incomplete IL-10R blockade or a generally minor contribution of IL-10 to Treg-mediated suppression in vitro. In summary, we demonstrate that ST2 expression identifies a highly activated, strongly suppressive Treg subset located in non-lymphoid tissues. The phenotype and function of these cells overlaps in many aspects with previously described effector Treg subsets. However, ST2 + Tregs stand out from these populations as they feature a Th2-biased phenotype already under homeostatic conditions. Additionally, these cells employ a mechanism based on IL-10 and increased TGFβ production and activation to mediate their enhanced suppressive function. Due to their specific capabilities, ST2 + Tregs may be suitable for targeted immunomodulatory therapies, e.g. to alleviate allergies or autoimmunity. First, we wanted to assess whether IL-33 signaling via the ST2 receptor is necessary for the development and maintenance of ST2 + Tregs present under homeostatic conditions. Therefore we isolated splenocytes from naive WT mice, and analyzed the frequency of ST2 + and ST2 - Foxp3 + CD4 + T cells(Fig 1A and S1A Fig). As reported previously a subpopulation of Tregs expressed the receptor for IL-33. Next, we determined the frequency and number of total Tregs in WT, ST2-deficient (Il1rl1 -/-) and Il33 -/-mice. We found that both the frequency and number of Tregs were largely comparable in the spleen, peripheral lymph nodes (pLN) and lung of these mice (Fig1B). Although a significantly elevated frequency of splenic Tregs was detected
Fig 1 . 1 Fig 1. ST2 + Tregs arise independently of IL-33 signals. Phenotype of Tregs in naive WT, Il1rl1 -/-and Il33 -/-mice: (A) ST2 and Foxp3 expression by splenic CD4 + T cells of one representative naive WT mouse; quadrant numbers indicate the average frequency ± SD in 4 mice. (B) Frequencies and total numbers of FoxP3 + Tregs in spleen (Spl), peripheral lymph nodes (pLN) and lung. (C) Frequency of ST2 expression in Tregs of spleen and lung. (D) Total number of ST2 + Tregs in spleen, pLN and lung. Fig 2A, 2C and 2D: Data are representative of at least 2 independent experiments. Fig 2B: Pooled data from 2 independent experiments, each with 4 mice per genotype. Bar graphs show the mean ± SD of at least 4 individual mice. Significance was tested using unpaired Student's t test. Asterisks indicate significance; all others non-significant. * p 0.05; p 0.01; * p 0.001. doi:10.1371/journal.pone.0161507.g001
4 + 4 Tregs was clearly increased in the ST2 + compartment, especially within lymphoid tissues (Fig 2D). Moreover, we analyzed the expression of transcription factors in the ST2 + and ST2 -Treg subsets. Both the Treg-key regulator Foxp3 as well as the Th2-associated transcription factor GATA-3 were elevated in ST2 + Tregs (Fig 2E and S3A Fig). In contrast, expression of T-bet, which can be induced in Tregs upon Th1-differentiating antigen encounter , was not altered (S3B Fig). Although specific to ST2 + Tregs, we found the expression of all of the aforementioned molecules to be independent of IL-33 (S3C and S3D Fig).
Fig 2 . 2 Fig 2. ST2 + Tregs preferentially home outside of secondary lymphoid organs and exhibit a highly activated phenotype. Flow cytometric analysis of the phenotype and frequency of WT ST2 + and ST2 -Foxp3 + Tregs in spleen, pLN, blood, lung, lamina propria of the small intestine (siLP) and colon (coLP): (A) Frequency of ST2 + Tregs (left) and MFI of the ST2 staining on the ST2 + Treg fraction (right). (B) MFI of chemokine receptor and α4β7 staining on ST2 + and ST2 -Tregs. (C) KLRG1 and CD103 expression in ST2 + (top) and ST2 -(bottom) Tregs from spleen; quantified frequencies from indicated organs (right). (D) Frequency of CD44 hi , CD62L lo and CTLA-4 + T cells within ST2 + and ST2 -Treg populations. (E) MFI of the Foxp3 staining (left) and geometric mean index of GATA-3 (right) in ST2 + and ST2 -Tregs. (F) Quantification of mRNA expression of the indicated genes from FACS-sorted ST2 + and ST2 -CD25 + Tregs from spleen and pLN ex vivo. mRNA expression normalized to Hprt endogenous control. (G) Frequency of ST2 + and ST2 -Tregs with IL-10 production capability as detected by GFP expression from B6. Foxp3 hCD2 xIl10 gfp reporter mice. Fig 2A: Data are representative of at least 2 independent experiments. Bar graphs show the mean ± SD of at least 5 biological replicates. Fig 2B: pooled data from 2 independent experiments with 3-5 biological replicates each. Bar graphs show the mean ± SD. Fig 2C-2E and 2G: Data are representative of at least 2 independent experiments. Scatter plots depict one mouse as individual dot with mean ± SD. Fig 2F: pooled data from 2 independent experiments. Significance was tested using unpaired Student's t test. * p 0.05; p 0.01; * p 0.001; non-significant (ns) p > 0.05. doi:10.1371/journal.pone.0161507.g002
Fig 3 . 3 Fig 3. ST2 + Tregs suppress CD4 + T cell proliferation more effectively than ST2 -Tregs in vitro. (A) Proliferation profiles of CellTrace-labelled WT CD25 -CD62L hi CD4 + responder T cells (Tresp) co-cultured with WT ST2 + (black) and ST2 -(grey) CD25 + Tregs during an in vitro suppression assay at day 4 of culture. T cells were stimulated by APCs and anti-CD3 antibody with (right column) or without (left column) the addition of recombinant IL-33. Treg:Tresp ratios are indicated (left). Percentage of divided cells and the division index (number in brackets) are shown in each histogram in the respective color. (B) Proliferation profile of Tresp cultured under the same conditions as in 3A but without Tregs, either with (grey) or without (black) the addition of anti-CD3 antibody. (C) MFI of the ST2 staining on all Tregs recovered from the cultures described in 3A. Data are representative of 2-3 independent experiments. doi:10.1371/journal.pone.0161507.g003
ST2+ Tregs express TGFβ and the Th2 cytokines IL-5, IL-13 and IL-10 Although IL-33 did not influence the suppressive capacity of ST2 + Tregs in vitro, it still increased the amount of ST2 expressed on their cell surface. To assess whether other functions are affected by the capacity of ST2 + Tregs to sense IL-33, we stimulated ST2 + and ST2 -Tregs in vitro in the presence or absence of this cytokine. We observed that the number of living cells was highest when ST2 + Tregs were cultured in the presence of IL-33 (Fig 4A). Thus, IL-33 affects either their proliferation or survival. Moreover, ST2 + Tregs produced more TGFβ, IL-5, IL-13 and IL-10 at the mRNA and protein level than their ST2 -counterparts, and the production of the Th2-associated cytokines IL-5 and IL-13 was vastly increased by IL-33 (Fig 4B and 4C, S4 Fig). GATA-3 expression was also significantly higher in ST2 + than ST2 -Tregs but unaffected by IL-33 signaling (Fig 4D). In contrast, IFNγ secretion was significantly lower by ST2 + than ST2 -Tregs. Almost no IL-4 was detectable in the supernatants of stimulated ST2 + and ST2 -Tregs with only minor differences between the groups. Hence, we conclude that IL-33 positively influences the expansion of ST2 + Tregs and promotes the production of the Th2 cytokines IL-5 and IL-13.
Fig 4 . 4 Fig 4. ST2 + Tregs express Th2 cytokines and suppress CD4 + T cell proliferation via IL-10 and TGFβ. (A-D) ST2 + and ST2 -Tregs from spleen and lymph nodes of WT mice activated in vitro by plate-bound anti-CD3/anti-CD28 antibodies in the presence of IL-2 with or without recombinant IL-33 for 60-70 hours: (A) Fold change in the number of viable Tregs upon IL-33 treatment. (B) Tgfb1 mRNA expression normalized to Hprt endogenous control. (C) Cytokine concentration in the supernatants as determined by cytometric bead array. (D) Geometric mean index of GATA-3 in stable ST2 + and ST2 -Tregs at the end of culture. (E) In vitro suppression assay with ST2 + and ST2 -Tregs as described in Fig 3 (Treg:Tresp ratio 1:5) with addition of blocking anti-IL-10R antibody or TGFβRI inhibitor. The relative division index indicates the fold increase in division of Tresp upon treatment. Division index of untreated Tresp was set to 1 in each group. (F) Quantification of mRNA expression of the indicated genes from sorted ST2 + and ST2 -CD25 + Tregs ex vivo. mRNA expression normalized to Hprt endogenous control. Fig 4A-4C, 4E and 4F, data pooled from 2-3 independent experiments each performed with 2 replicates per condition. Fig 4D is representative of 2 independent experiments with at least 2 replicates per condition each. Bar graphs show the mean ± SD. Significance was tested using unpaired Student's t test. * p 0.05; p 0.01; * p 0.001; non-significant (ns) p > 0.05. doi:10.1371/journal.pone.0161507.g004
PLOS ONE | DOI:10.1371/journal.pone.0161507August 22, 2016
|
10.3390/nu14153090
|
cc-by
| null | null |
openalex
|
The association of hyperinflammation and hyperferritinemia with adverse outcomes in SARS-CoV-2-infected patients suggests an integral role for iron homeostasis in pathogenesis, a commonly described symptom of respiratory viral infections. This dysregulated iron homeostasis results in viral-induced lung injury, often lasting long after the acute viral infection; however, much remains to be understood mechanistically. Lactoferrin is a multipurpose glycoprotein with key immunomodulatory, antimicrobial, and antiviral functions, which can be found in various secreted fluids, but is most abundantly characterized in milk from all mammalian species. Lactoferrin is found at its highest concentrations in primate colostrum; however, the abundant availability of bovine-dairy-derived lactoferrin (bLf) has led to the use of bLf as a functional food. The recent research has demonstrated the potential value of bovine lactoferrin as a therapeutic adjuvant against SARS-CoV-2, and herein this research is reviewed and the potential mechanisms of therapeutic targeting are considered.Introduction Iron is vital to living cells because of the essential roles it plays in various biological systems such as cytochromes, oxygen binding molecules, and enzymes. Not only is iron necessary for proper cellular function, but its concentration must be carefully controlled, as both high and low iron levels can lead to cell injury and death [1, 2] . The maintenance of iron homeostasis is primarily mediated through regulating dietary iron absorption by enterocytes and the release of recycled iron from macrophages. Iron homeostasis can be significantly impacted by infection and the host immune response. For example, severe systemic iron overload can result in an increased susceptibility to infection and the secretion of proinflammatory cytokines can disrupt iron homeostasis [3, 4] . During infection and inflammation, including viral infections, macrophages become iron-overloaded . To counter the viral disruption of iron homeostasis, the multifunctional glycoprotein lactoferrin has been proposed as a bioactive therapeutic, including most recently for the treatment of SARS-CoV-2 infections . Herein, we review the dynamic interactions between viral infection, iron homeostasis, and inflammation, focusing specifically on the potential for lactoferrin as a therapeutic adjuvant for the treatment of COVID-19, the disease caused by infection with the SARS-CoV-2 virus.
Iron Homeostasis and Viral Infection Dietary iron enters the body through enterocytes lining the intestinal wall via the enzymatic action of the duodenal cytochrome B, which catalyzes the reduction of Fe 3+ to Fe 2+ , and the divalent metal transporter 1, which binds Fe 2+ . Ferrous iron is then released into the plasma through ferroportin . Ferrous iron is converted to the ferric form by either the hephaestin or ceruloplasmin ferroxidases, found in the small intestine, or hepatocytes and macrophages, respectively, and bound to serum transferrin (Tf) for transportation and storage in cytosolic ferritin . The regulation of iron homeostasis is dependent on the diet-mediating hormone hepcidin, which binds ferroportin, leading to internalization and degradation . Increased iron in the serum leads to the increased expression of hepcidin, with concurrent retention of iron in macrophages and reduced absorption of dietary iron. Conversely, low levels of iron in the serum will trigger the suppression of hepcidin expression and increased release of intracellular iron . Hepcidin is also an acute-phase protein, synthesized in response to inflammatory conditions through IL-6 , thereby linking iron homeostasis and inflammation through the regulation of hepcidin. The mechanistic relationship between viral infection and iron homeostasis is not fully understood; however, viral infections can alter iron homeostasis (see Drakesmith and Prentice for an excellent review ). For example, inflammatory cytokines induced in viral infections can impact cellular and systemic iron concentrations by inducing hepcidin synthesis, blocking ferroportin assembly and transferrin release . Influenza A, which uses the transferrin receptor protein (TfR1) to enter the cell, enhances the expression of the proinflammatory cytokine IL-6 (Figure 1 ) . This increased production of IL-6 was reduced three-fold in the presence of the hydroxyl radical scavenger dimethylthiourea. During viral infections, particularly chronic viral hepatitis, circulating free iron has been detected . Whether this circulating free iron is the result of hepatic cell death, the release of free iron from dying macrophages with iron overload, or a combination of both remains debatable. Because free iron can induce reactive oxygen species to further perpetuate the inflammatory response, this finding strengthens the idea that iron levels above the homeostatic parameters could potentially cause patients with viral infections to have less favorable outcomes due to an excessive inflammatory response . While there are many different ways in which viruses can impact iron metabolism and alter iron homeostasis, the findings from human immunodeficiency virus (HIV-1) infection provide some insight. HIV-1 has evolved many mechanisms to support host cell iron retention to the benefit of long-term viral survival and replication. For example, the myristolated protein Nef of HIV-1 downregulates the macrophage major histocompatibility complex (MHC-1b) expression of the homeostatic iron regulator (HFE) protein. HFE is a membrane protein that helps regulates the circulating iron uptake by regulating the interaction of the transferrin receptor (TfR) with transferrin (Tf). Pathologically low levels of HFE result in ferritin iron accumulation in macrophages, which are inversely correlated with survival times . Additional predictors of mortality in HIV-1 infection include genetic polymorphisms in iron regulatory genes, such as the SLC11A1 gene, encoding the natural-resistance-associated macrophage protein-1 (Nramp1), and the HP gene, encoding the free-hemoglobin scavenger haptoglobin. Nramp1 is hypothesized to act as an iron gatekeeper in macrophages and is key to the host resistance to infection. SLC11A1 gene mutations have been associated with increased susceptibility to infectious diseases, as well as diseases of chronic inflammation; however, the iron homeostasis contribution of Nramp1 and haptoglobin during HIV-1 infection remains an area of debate . Finally, it has been demonstrated that the iron chelator deferoxamine decreases viral replication in HIV-1 infections by impacting the irondependent viral replication . In addition to HIV, other viruses such as cytomegalovirus, members of the arenavirus family, and members of the picornaviridae family have been shown to be linked to disrupted iron homeostasis and dysregulated iron metabolism during infection [12, 19] .
Lactoferrin as a Therapeutic Adjuvant in Respiratory Viral Infections
Bovine Lactoferrin: A Multifunctional Glycoprotein Increasingly, the bioactive value of dairy products has led to an expansion of dairyderived functional foods on the market. Whey is derived from the cheese making process and used to be discarded as waste; however, the characterization of bioactive proteins in whey has revived interest in the market value of whey and its derivatives as nutraceuticals . One such bioactive protein found in whey is the iron-binding glycoprotein lactoferrin. Lactoferrin (Lf) is produced by all mammals and can be found in most secretory fluids such as colostrum, milk, saliva, and tears , as well as neutrophil secretory granules . At ~80,000 Da, lactoferrin is a larger member of the transferrin family of non-heme, iron-binding glycoproteins. With two structural lobe domains, Lf can bind two ferric ions (Fe 3+ ) with high affinity within each lobular cleft (Kd = 10-20 M). Depending on whether Lf is iron-bound or unbound, the protein assumes either the open form (apo-lactoferrin) or closed form (halo-lactoferrin) . The two lobes are connected by a short peptide, which forms a 3-turn α-helix that can be cleaved to produce the lactoferrin peptide derivatives lactoferampin and lactoferricin, which have been shown to have antimicrobial properties . Originally thought to function as an iron transporter, the innate immune-modulating characteristics of Lf are increasingly appreciated, including inhibiting neutrophil priming by bacterial lipopolysaccharide (LPS), enhancing neutrophil adherence to endothelial cells, and modulating inflammation by amplifying apoptotic signals (referred to as ferroptosis) . Dairy-derived bovine lactoferrin (bLf) is generally recognized as safe (GRAS) by the FDA and is commonly used as a nutraceutical dietary supplement . Of the total milk proteins found in bovine milk, 20% are whey protein, with 1.5% of the whey protein consisting of bLf. In contrast, human milk consists of 60% whey proteins, with an average of 2.5% consisting of human lactoferrin (hLf) [27, 28] . Despite the difference in lactoferrin abundance between humans and bovines, of all lactoferrin-producing non-primates, bLF is the most similar structurally to the lactoferrin that is endogenously produced by humans . However, some distinctions between human lactoferrin (hLf) and bovine lactoferrin (bLf) have been demonstrated. For example, bLf is proposed to bind up to four times the amount of iron bound by hLf , and hLf contains multiple unique steroid response elements . Additionally, differences in Lf distribution exist between species, with bLf being more abundant in saliva and produced by bronchopulmonary structures such as the serous and mucous cells of the bronchial glands, while hLf has been shown to be absent from lung alveoli [21, 31] . These species-specific differences in the chemical properties and physiological distribution may indicate functional distinctions; however, both hLf and bLf exhibit an array of characteristics during infection. During viral infection, Lf directly impacts the host immune response through the rebalancing of iron homeostasis, the modulation of inflammation, and the promotion of antiviral gene expression . For example, the oral administration of bLf has been shown to reduce levels of IL-6 systemically, leading to decreased hepcidin and increased ferroportin . As described above, this leads to the increased release of intracellular iron stores and dietary uptake of iron to counter inflammation-induced anemia. More passively, Lf directly binds free iron, thereby limiting the iron-dependent inflammatory processes in tissues . There are many mechanisms by which Lf reduces inflammation, including both active and passive processes. For example, the in vitro culture of human macrophages with bLf has also been shown to play a role in shifting LPS-stimulated, pro-inflammatory M1 macrophages to the M2 anti-inflammatory phenotype . Whether this shift is through the direct modification of gene expression or through more passive means of metabolic immunomodulation remains to be determined; however, multiple studies have shown that both hLf and bLf are transported into the nucleus and can alter gene expression (Figure 2B ). Indeed, Lf is proposed to function as a transcription regulator and to directly inhibit the expression of pro-inflammatory cytokines . For example, Ashida et al. demonstrated that differentiated intestinal epithelium-like Caco-2 cells bound and internalized endogenously expressed Lf at the apical membrane, eventually localizing Lf to the nucleus . In another in vitro study using peripheral blood-derived human mononuclear cells (PBMCs), bLf was rapidly brought into the nucleus during monocyte cellular differentiation, correlating the differentiation with bLf reaching the nucleus . Using primary bronchial cells derived from cystic fibrosis patients, Valenti et al. demonstrated that the addition of bLf during infection reduced inflammation through decreased pro-inflammatory cytokine expression and increased anti-inflammatory IL-10 expression (Figure 2B ). Interestingly, the same study also asserted that bLf only altered the expression of cytokines in infected cells and not in uninfected cells . Finally, using human umbilical vascular endothelial cells (HUVECs), Kim et al. demonstrated in vitro that hLf interfered with the TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) by downregulating ICAM-1 expression through a DNA-binding-dependent manner (Figure 2B ). Clearly, there is mounting evidence suggesting that Lf, of both bovine and human origin, can actively modulate gene transcription, impacting both iron homeostasis and the inflammatory process.
Antiviral Activity of Lactoferrin The antiviral activity of lactoferrin has been demonstrated both in vitro and in vivo (summarized in Table 1 ). This antiviral property was first demonstrated in mice infected with the polycythemia-inducing strain of the friends virus complex (FVC-P). For this study, mice were intravenously dosed with hLf, which prolonged their survival rates and decreased the viral load of FVC-P . Lf is able to inhibit viruses, in part, through iron chelation ; however, subsequent studies indicated that the antiviral mechanism of lactoferrin is much more complex. Due to its cationic features and its ability to bind iron at a low pH, Lf is able to interact with many surface molecules and ions, including heparan sulfate proteoglycans, cell receptors, and enveloped viral particles, leading to the disruption of viral maturation and inhibition of immunomodulatory activation [2, 40, 41] . Table 1 . Summary of antiviral studies with lactoferrin. In silico, in vitro, and clinical trials utilizing human, bovine, or other sources of lactoferrin are outlined below with the source of lactoferrin, route of administration (if in vivo), and summary of the relevant results from the main text.
Author (Year) [Citation] Model Lactoferrin Source Route (Dose) Brief Results
Sinopoli et al. (2022) Systemic Review NA Systemic review of clinical trials using orally administered Lf for the treatment of viral infections.
Marchetti et al. (1996) Primate In vitro hLf bLf Lf inhibits HSV1 absorption with bLf showing better efficacy than hLf.
Lu et al. (1987) Murine In vivo hLf i.p. hLf shown to have protective effects against the polycythemia-inducing strain of the friend virus complex in mice.
Marchetti et al. (1998) Primate In vitro bLf The antiviral activity of Lf appears to correlate with the degree of its metal binding and saturation.
Yi et al. (1997) In vitro bLf hLf Demonstrates interaction of Lf and HCV envelope proteins.
Ishii et al. (2003) Human Clinical bLf oral (0.6 g/day) Increased IL-18 with oral bLf supplement in chronic HCV patients.
Okada et al. (2002) Human Clinical bLf oral (1.8-7.2 g/day) bLf use in chronic hepatitis C patients is well tolerated.
El-Ansary et al. (2016) Human Clinical bLf oral (0.5 g/day) Increased CD4, CD8, CD137, and CD56 levels with bLf supplementation in chronic HCV patients
Ueno et al. (2006) Humans Clinical bLf oral (1.8 g/day) Oral Lf has a negligible impact on viral load when taken orally by patients with chronic HCV.
Tanaka et al. (1999) Humans Clinical bLf oral (1.8-6 g/day) Lf could be used as an anti-HCV adjuvant therapy with the potential to help treat chronic hepatitis.
Hirashima et al. (2004) Human Clinical bLf oral (9.0 g/day) Lf did not increase the response rate or prevent relapse after discontinuing interferon in chronic HCV patients.
Ishibashi et al. (2005) Human Clinical bLf oral (0.6 g/day) This study failed to demonstrate that Lf in combination with antiviral therapy provided additional benefit to chronic HCV patients.
Kaito et al. (2007) Human Clinical bLF oral (3.6 g/day) Lf was shown to increase the effectiveness of interferon and ribavirin therapy in chronic HCV patients.
Konishi et al. (2006) Human Clinical bLf oral (3.6 g/day) Decreased ALT levels and plasma 8-isoprostane in chronic HCV patients.
Ochoa et al. (2013) Human Clinical bLf oral (0.5 g/day) Decreased duration and symptoms in norovirus patients.
Egashira et al. (2007) Human Clinical bLf oral (100 mg/day) Decreased frequency and duration of symptoms in rotavirus patients.
Zuccotti et al. (2007) Human Clinical bLf oral (3 g/day) Observed decline in viral load during bLf administration in HIV patients.
Mirabelli et al. (2020)
Human
Miotto et al. (2021) In silico hLF Computational modeling indicated that Lf blocks SARS-CoV-2 infection through competitive binding with the spike protein.
Piacentini et al. (2022) In silico hLf Lf binds to ACE2 receptor and not SARS-CoV-2 spike protein RBD.
Campione et al. (2021) Human Primate In vitro bLf Lf effective antiviral against SARS-CoV-2 infection in vitro.
Cutone et al. (2022) Human In vitro bLf Preincubation with bLf inhibited SARS-CoV-2 binding and pseudovirus entry into epithelial and macrophage-like cells, reduced inflammatory response, and increased gene expression associated with iron homeostasis.
Serrano et al. (2020) Human Clinical bLf oral (20-30 mg/day) Improvement in reported symptoms in mild to moderate COVID-19 patients.
Campione et al. (2020) Human Clinical bLf oral (1 g/day) Decreased time to negative molecular test and duration of symptoms in COVID-19 patients
Algahtani et al. (2021) Human Clinical bLf oral (200-400 mg/day) No statistical difference between treatment and non-treatment groups, but trends in symptom improvement and blood biomarker profile observed.
Rosa et al. (2021) Human Clinical bLf oral (200-1000 mg/day) Reduced time to negative molecular SARS-CoV-2 test, reported reduction in symptoms of COVID-19 patients of advanced age. Abbreviations: NA = Not applicable, bLf = bovine lactoferrin, hLf = human lactoferrin, i.p. = intraperitoneal. Despite this diverse functional range, certain factors have been shown to impact Lf's efficacy in limiting viral infection. For example, the evidence from multiple in vitro studies suggests that the antiviral effects of Lf are particularly effective early and are mediated primarily through the prevention of viral entry into host cells [2, 52, 53] . In addition, the antiviral efficacy may be impacted by subtle differences between species, with bLf reported to exhibit higher antiviral activity than hLf [2, 42] . For example, Marchetti et al. showed that both hLf and bLf have the ability to interfere with herpes simplex virus-1 (HSV-1) infection by binding heparin sulfate proteoglycans and LDL receptors (Figure 2A ); however, bLf was shown to be more potent in this case . The specific biochemical or structural differences that contribute to Lf's functional divergence between primates and bovines remains to be established; however, the species of origin may impact Lf's antiviral efficacy. The growing consensus also indicates that Lf's antiviral activity levels are not identical between infecting virus types 71] . While the above studies indicate that Lf's antiviral activity is mediated through the competitive inhibition of viral binding and entry into the host cell, other studies have demonstrated Lf's inhibition of viral-infection-mediated post viral attachment. For example, in an in vitro study examining both human and bovine Lf inhibition of poliovirus replication, Manchetti et al. demonstrated that both hLf and bLf prevented the viral replication of poliovirus, regardless of whether iron, magnesium, or zinc was bound; however, the post-attachment inhibition of viral infection was dependent on zinc-bound Lf, with the levels of zinc and degree of inhibition being directly correlated . Another in vitro study examined the role of bLf in limiting rotavirus infection and found that the removal of sialic acid enhanced the anti-rotavirus activity of bLf, concluding that the mechanism of viral suppression in rotavirus infection is not dependent on the competitive inhibition of viral attachment, as shown for HSV infection . In some circumstances, Lf may exert a variety of antiviral mechanisms to prevent infection. In both in vitro and in vivo studies using pull down assays, the role of Lf in hepatitis-C virus (HCV) infection has shown that Lf directly binds the HCV envelope protein E2 (Figure 2A ), rather than competitively inhibiting HCV attachment to the host cell [44, 47] . In addition, further in vitro studies on HCV and Lf showed that HCV leads to elevated intracellular iron stores. This elevation of iron levels increased the susceptibility to infection by the virus, suggesting that the iron sequestering role of Lf also helps limit the viral pathogenesis . Lf has also been shown to prevent hepatitis-B virus (HBV) infection; however, in contrast to HCV infection, the proposed mechanism is through the competitive binding of the glycosaminoglycan receptor (Figure 2A ), suggesting that Lf antiviral mechanisms may vary even within closely related viruses. These promising in vitro and in vivo studies, as well as the GRAS status of Lf, have led to an increased interest in the clinical use of Lf in the treatment of viral infections (systematically reviewed by Sinopoli et al. ). Using a standardized approach [72, 73] , Sinopoli et al. identified 27 records investigating the use of orally administered Lf to prevent or manage viral infections (Table 1 ). The viral infections studied included HCV, HIV, rotavirus, norovirus, and SARS-CoV-2, and ranged from non-randomized to randomized, dose-response, and controlled trials. Minimal side effects were identified across all studies, and the treatment impacts varied from no observed differences to significant impacts on the viral load, immune response, and reported symptoms. For example, the management of chronic HCV patients with oral bLf resulted in significant decreases in alanine transaminase and plasma 8-isoprostane and significantly increased IL-18, CD4, CD8, CD137, and CD56 levels 55] . For norovirus, rotavirus, and SARS-CoV-2, decreased duration and severity of symptoms were observed [56, 57, 65, 67] . Finally, decreases in the viral load and duration of viral detection were observed for both HIV and SARS-CoV-2 [58, 65] . While these studies provide promising results, establishing oral Lf as a therapeutic option for the management of viral infections will require larger and more robust clinical trials.
Lf as a Therapeutic Adjuvant in COVID-19 Since the emergence of the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it has effectively spread across the globe, with more than 529 million people confirmed to have been infected and more than 6.3 million deaths by the spring of 2022 (the Johns Hopkins Coronavirus Resource Center: https://coronavirus.jhu.edu, accessed on 1 June 2022). Even with the successful development of vaccines, much of the population remains susceptible to the virus due to such factors as a lack of access to vaccines, hesitancy towards vaccination, and the emergence of viral variants. In addition, the long-term impacts of COVID-19 have led to the emergence of chronic pathologies referred to as "ongoing symptomatic COVID-19" for symptoms lasting between four and twelve weeks post-acute infection and "post-COVID-19 syndrome" for symptoms lasting longer than twelve weeks . With the continued spread of COVID-19, and despite the development of SARS-CoV-2 vaccines, there remains the need to develop novel therapeutics for both acute and chronic COVID-19. Disrupted iron homeostasis has been associated with worse outcomes in viral infections, as previously mentioned, and the same is seemingly true for patients with COVID-19. The accumulating evidence suggests that iron chelation therapy is a promising adjuvant therapy for COVID-19 . While the mechanism of action is still under debate, COVID-19 is known to be associated with increased levels of the proinflammatory factors IL-1β, IFNγ, IP-10, and MCP-1, the expression of which is sensitive to iron homeostasis . Additionally, macrophages are presumed to be infected by COVID-19, and increased iron storage in macrophages may favor viral replication . More recently, Cutone et al. demonstrated in both enterocytes and macrophages that the SARS-CoV-2 spike protein can induce the dysregulation of major iron-handling proteins, including the downregulation of ferroportin, DMT-1, and hephaestin and the upregulation of TfR1 . Patients with severe COVID-19 infections tend to have elevated ferritin and IL-6 levels, with the serum ferritin levels demonstrated to be twice as high in non-survivors . High serum ferritin levels are linked to cardiovascular events in addition to inflammatory pathologies, and cardiac damage is a common outcome of severe COVID-19 . Ferroptosis, a form of programmed cell death mediated by iron, is initiated by alkoxyl radicals produced by ferrous iron . Ferroptosis has been shown to be linked to inflammation, as it involves multiorgan inflammatory pathways in the liver, kidney, heart, and lungs . Observed in both adult COVID-19 and the pediatric multisystem inflammatory syndrome in children (MIS-C), the inflammatory damage of the host tissue can occur in the most severe cases of SARS-CoV-2 infection . Interestingly, ferroptosis is also associated with ageusia and anosmia (loss of taste and smell, respectively). Hypogeusia, a reduction in tasting ability, and reduced olfactory function were associated with iron deficiency in patients [85, 86] . Just as with iron deficiency and ferroptosis, COVID-19 has also been shown to be associated with anosmia and ageusia . Another known risk factor and major concern for COVID-19 patients is the disruption of the coagulation cascade. Iron plays a complex role in coagulation, extending the clotting of plasma by interacting with proteins of the coagulation cascade, while increasing the risk of thrombosis by precipitating plasma proteins . COVID-19 patients have already been shown to be at an increased risk for thromboembolism. In fact, the preliminary reports on COVID-19 patients show that the patients can often present with thrombocytopenia, elevated D-dimer levels, prolonged prothrombin time, and disseminated intravascular coagulation, along with coagulation cascade abnormalities . Other studies have touched on the inflammatory and procoagulant condition of COVID-19 patients, which is especially prominent in patients with less-favorable outcomes . One study with five patients under the age of 50, all with confirmed SARS-CoV-2 infection, reported that each patient experienced new-onset large-vessel strokes . Similarly, another study examined patients in a COVID ICU, where clinically significant coagulopathy and multiple infarcts were seen . From an excessive proinflammatory state to multiorgan oxidative damage to anosmia and ageusia, the pathologies associated with COVID-19 indicate that beyond a characteristic hyperferritinemia, this disease exhibits many features of the systemic dysregulation of iron homeostasis.
Antiviral Activity of Lactoferrin against SARS-CoV-2 Because of its ability to modulate viral infection, iron homeostasis, and inflammation, Lf warrants consideration as an adjuvant therapy in the treatment of COVID-19. In particular, the use of dairy-derived lactoferrin has been shown to be efficacious in vitro and in vivo, can be economically produced in large quantities, and is well tolerated in clinical trials. Over the course of the pandemic, suggestive data supporting this therapeutic approach have emerged. Mirabelli et al. used quantitative high-content morphological profiling coupled with AI-based machine learning to screen 1425 FDA-approved compounds and clinical candidates, and found bovine lactoferrin to be an effective inhibitor of SARS-CoV-2 infection. The mechanistic basis for this inhibition was not clear; however, they showed that Lf blocks SARS-CoV-2 viral entry and rescues infection up to 24 h post-infection in a dosedependent reduction in viral replication. In addition, they observed elevated mRNA levels of IFN-β and associated IFN genes (ISG15, MX1, Viperin, and IFITM3) in Huh7 cells . Subsequently, Lf was shown in vitro to inhibit SARS-CoV-2 infection and replication in Caco-2 intestinal epithelial cells, with an associated increased expression of antiviral genes (IFNA1, IFNB1, TLR3, TLR7, IFIH1, IRF3, IRF7, and MAVS) and decreased expression of the anti-inflammatory cytokine TGFB1 . More recently, Wotring et al. screened H1437 human lung cells cultured in vitro and pre-treated with a series of dairy products against several SARS-CoV-2 emergent variants of concern (B.1.1.7, B.1.351, and P.1), finding that the antiviral efficacy of the dairy products was concentration-dependent on bLf . These in silico and in vitro results further support the potential of bLf as a nutraceutical protein for treating SARS-CoV-2 infection. How Lf can inhibit SARS-CoV-2 viral entry is currently under investigation, but the likely mechanisms include directly binding host cell factors (competitive inhibition) or directly binding viral particles (Figure 2 ), as previously described for other viruses. While bLf has been shown to directly bind viral structural proteins such as S, M, and E , a number of studies have suggested that Lf may directly interfere with the viral entry into host cells through the competitive inhibition of viral binding to glycan attachment factors. The two key classes of glycans include the sialic acid glycans and glycosaminoglycans, such as heparan sulfate. While the angiotensin-converting enzyme (ACE2) receptor is a well-established binding target for SARS-CoV-2 , glycans are proposed to initiate contact with viral particles, shuttling the virus to the target binding receptor on the host membrane and facilitating viral engulfment . Both sialic acid and heparan sulfate glycan receptors have been shown to be involved in SARS-CoV-2 infections , and Lf may block SARS-CoV-2 viral entry by binding these accessory targets to viral entry. The computational modeling of molecular interactions demonstrated putative regions on the surface of Lf that could potentially competitively inhibit the viral attachment to sialic acid and the complementary surface structure on the SARS-CoV-2 spike protein, potentially allowing Lf to compete with ACE2 receptors for binding directly to viral particles . More recently, Piacentini et al. used bilayer interferometry and latex nanoparticleenhanced turbidity to measure both the kinetic and thermodynamic parameters of hLf binding to the SARS-CoV-2 receptor-binding domain (RBD) and the ACE2 receptor. Their results indicated that hLf binds the ACE2 receptor but not RBD at physiological concentrations of hLf. Interestingly, they found that Lf bound the ACE2 receptor ectodomain, indicating that hLf may competitively inhibit RBD binding by either directly interfering with the ACE2-RBD interaction or by binding an ACE2 receptor region far from the RBD site, triggering conformational changes of the ACE2 receptor and inhibiting RBD binding . In contrast, Cutone et al. recently showed that both hLf and bLf do interact with the SARS-CoV-2 trimeric form of a full-length spike protein, even with the introduction of point mutations observed in recent viral variants . Clearly, further studies are warranted; however, these in vitro results are promising, especially given the continued efficacy of Lf when challenged with newly emergent viral variants.
Clinical Evidence of Lactoferrin Efficacy in COVID-19 Patients Clinically, the evidence for the use of Lf as a therapeutic adjuvant for SARS-CoV-2 infection is limited but rapidly increasing. Campione et al. performed a small (n = 32) human trial on bLf embedded in liposomes for oral and nasal administration in asymptomatic to mild symptom COVID-19 cases. This treatment resulted in early viral clearance (15-30 days) along with decreases in IL-6, D-Dimer, and ferritin serum levels. The in silico modeling suggested the direct binding of Lf to S protein in the study. While these results are indeed promising, it is important to note that there was no placebo control group in the study, nor was it a double-blind clinical trial . Another pilot clinical trial (n = 54) using oral bLf for mild to moderately symptomatic COVID-19 patients was randomized and assayed for dose-dependent responses. While no statistically significant differences between the control and treatment groups were observed, some interesting trends were noted. For example, the reported symptoms of fever, dry cough, diarrhea, headache, loss of sense of taste or smell, and fatigue were improved with Lf treatment. Increased serum hemoglobin, lymphocyte count, and platelet count levels were detected at the highest dose of Lf (200 mg, twice daily), while the levels of C-reactive protein declined . Finally, in a retrospective study on asymptomatic, paucisymptomatic, and moderately symptomatic COVID-19 patients in home-based isolation, Rosa et al. compared Lf-treated (n = 82) to Lf-untreated (n = 39) patients using bLf unloaded in liposomes. The median number of days to negative molecular test for the SARS-CoV-2 virus was lower in bLf-treated patients compared to non-treated patients (15 vs. 24 days, respectively); however, the times to symptom resolution did not differ significantly, with the exception of a subcohort of individuals of advanced age . While these preliminary clinical trials are promising, much larger randomized studies are needed to establish the clinical value of Lf supplementation. Finally, each of these clinical trials utilized oral Lf supplementation (with the exception of Campione et al. , who also used a nasal spray of liposomal Lf). Orally administrated Lf is subject to digestion by gastrointestinal proteases and can be absorbed as amino acids via facultative diffusion or active transport; however, Lf given orally interacts with receptors on gut epithelial cells and is likely to have systemic effects through signaling pathways. While Wotring et al. did assay the effect of lactoferricin f17-41, which is produced during digestion, against SARS-CoV-2 infection in vitro, they found this bLf derivative to be less effective than whole bLf. Clearly, much remains to be understood about Lf as a functional dietary supplement in general and as a therapeutic adjuvant against SARS-CoV-2 specifically.
Conclusions In conclusion, iron homeostasis is important for health, as iron levels play key roles during viral infections. Bovine lactoferrin's ability to modulate iron levels both systemically and locally, to mediate inflammatory processes, and to directly inhibit viral entry into host cells make it a potential therapeutic option for patients infected with SARS-CoV-2. As described above, bLf is structurally and functionally similar to hLf, can be effective when administered orally, is easily tolerated, and can be economically produced. These characteristics of dairy-derived bLf make it a viable candidate for further studies, specifically as a dietary supplement that is accessible to under-resourced populations impacted by the SARS-CoV-2 pandemic. While in many parts of the country the pandemic is still in the acute phase of its spread, Lf is also likely to have long-term value as we enter the post-vaccination phase of the pandemic. Approximately 30% of COVID-19 patients are reporting persistent symptoms lasting longer than nine months, despite having resolved the acute phase of their illness . The characteristics of post-acute sequelae of COVID-19 (PASC), otherwise referred to as long COVID, include fatigue, muscle weakness, insomnia, palpitations, chronic rhinitis, dysgeusia, chills, sore throat, and headache . Many of these symptoms are associated with the chronic, systemic inflammation and dysregulation of iron homeostasis, targets for which bLf is an established therapeutic option. While much remains to be understood about the therapeutic benefits of Lf as an adjuvant for SARS-CoV-2, the potential benefits of further investigations are likely to yield results both in the short-term pandemic surge and the longer-term outcomes of persistent symptoms in PASC patients. As the SARS-CoV-2 pandemic shifts towards endemic and potentially seasonal patterns of emergence, more clinical trials that examine the efficacy of oral bLf as a prophylactic for immune priming, a therapeutic adjuvant in acute infection, and a longterm nutraceutical for symptoms of PASC will provide the evidence needed to add bLf to our therapeutic toolbox in the fight against emerging viral pathogens such as SARS-CoV-2. Figure 1 . 1 Figure 1. Examples of iron homeostasis and viral infection. (A) Human immunodeficiency virus (HIV-1) blocks the homeostatic iron regulator (HFE) to increase iron accumulation in macrophages. (B) Influenza A binding of the transferrin receptor 1 (TfR1) promotes the expression of IL-6 and hepcidin degradation of ferropotin (FPN), also leading to intracellular iron retention and the promotion of viral replication.
Figure 2 . 2 Figure 2. Lactoferrin and viral infection. (A) Lactoferrin (Lf) can directly prevent viral entry through preventing viral engagement with host cell target receptors, such as hepatitis B virus (HBV) binding to glycosaminoglycan (GAG), hepatitis c virus (HCV) binding to cluster of differentiation 81 (CD81), and herpes simplex virus type 1 (HSV-1) binding to low-density lipoprotein receptor-related protein 1 (LRP1). (B) Lf uptake through the transferrin receptor (TfR) by host cells promotes the expression of antiviral genes and the inhibition of proinflammatory genes. (C) The proposed Lf mechanism in SARS-CoV-2 infection includes blocking viral binding to accessory target molecules, such as heparan sulfate proteoglycan (HS-PG) and sialic acid glycoprotein (SIA-PG), (D) direct binding to viral particles, and (E) the competitive inhibition of viral binding to the angiotensin-converting enzyme (ACE2) receptor. Additional abbreviations include: TGFB = transforming growth factor beta; IRF = interferon regulatory transcription factor; IFN = interferon; TLR = toll-like receptor; ICAM = intracellular adhesion molecule; IL = interleukin; TSLP = thymic stromal lymphopoietin.
activity against the human norovirusWotring et al. (2022) Human In vitro bLf Dairy product efficacy in inhibiting SARS-CoV-2 infection was dependent on Lf concentration; bLf retained efficacy against SARS-CoV-2 viral variants of concern.
Table 1 . 1 , bovine, and sheep Lf prevent HCV entry into cells by binding the virus; camel Lf was most effective. Cont. Marchetti et al. (1999) Primate In vitro bLf Suggests bLf plays a role in altering viral infection, particularly in the gut, through the inhibition of certain steps of viral infection. Superti et al. (2001) Primate In vitro bLf bLf inhibits rotavirus through a different mechanism than the previously reported for HPV. El-Fakharany (2013) Human, camelHara et al. (2002) Human In vitro hLf bLf camel Lf sheep Lf Human In vitro bLf hLf Lf inhibits HBV infection in vitro.
|
10.1192/pb.12.10.453
|
cc-by
| null | null |
openalex
|
difficult in long-term dynamic therapy, initial goals often changing in the course of therapy, and symp toms which were originally troublesome becoming better tolerated, even if still present. There is also the problem of patients vanishing after a course of psychotherapy before enough time has elapsed to make it clear whether or not significant changes have been maintained over time. This makes it difficult to evaluate success or failure of therapy with any accuracy and must be remedied if we are to make the best use of our limited resources. There is, indeed, a lack of training in helping with psycho-sexual problems. The availability and the type of training seems very haphazardly spread throughout the country, according to the where abouts of consultants with an interest in this field and the time to run clinics and supervise juniors. At present, I am one of the first two doctors to be taking the course run by the Marriage Guidance Council (Relate) on sexual dysfunction which offers a thorough training in behavioural methods of treat ment. Perhaps this or other courses might be avail able to a larger number of psychotherapy trainees in the future. On the question of personal therapy, it may depend on what sort of psychotherapy the trainee intends to practise as to whether personal therapy is considered essential or not. I have known one or two excellent dynamic therapists who have had no therapy themselves, but, particularly in this field, it is all too easy to over-identify with patients or to fail to understand how one's own prejudices and ideas affect them if one has had no experience of personal therapy. My own view is that I could not have begun to understand what my patients go through both in and between sessions had I not undergone the experi ence myself. Norman Macaskill1 in his review of the literature on personal therapy for therapists con cludes that there is no evidence that personal therapy for the therapist has a positive effect on outcome of therapy with patients, although there is evidence to suggest that the emotional health of the therapist is of importance in outcome. More research, he concludes, is essential in this area. Another issue involved in training future psycho therapy consultants is the supervision of supervision, when the trainee first starts supervising other junior staff, individually or in groups. Supervision is a skilled activity which needs to be learnt and therefore itself should be supervised by more experienced staff; this, I believe, is not standard practice at present. The last point deserving of attention is that raised by Clifford Yorkc.: He believes that psychotherapy consultants should not just be available to assess, treat and supervise but should be "teaching psycho analysis as a theory of mind (as opposed to a method of treatment)". Training in this small but pervasive subspeciality should involve developing the ability to communicate clearly with others about what one is doing and educate them when necessary in the under lying theoretical basis of one's work, rather than pre serving the mystique which has sometimes been attached to the practice of psychotherapy. As a psychiatrist who deals mainly with the elderly, but with over 20 years experience of using virtually one benzodiazepine -Chlordiazepoxide -1 find publications of this nature an inhibiting and constraining influence on good psychiatric practice.
S. HARTLAND Chlordiazepozide is a very good drug with vir tually no side effects. It is also helpful to know that in a dose of lOmgs it has little effect on psychomotor performance. A double-blind field trial with police drivers in Basle showed that it had no significant impairment of driving performance.' In my experience, the risk of dependence has not been a problem but this aspect of the benzodiaze pines must be taken into account in conjunction with the fact that very many patients have benefitted from taking them. Currently, I am concerned about patients who are suffering from the distressing symp toms of anxiety because of ineffective treatment by https://doi.org/10.1192/pb.12.10.453 Published online by Cambridge University Press inferior drugs, low-dosage phenothiazines and ßblockers. In the life history of a new drug there is a recognised cycle, its release with considerable hype and exaggerated expectation, the recognition of problems and disadvantages, and its eventual role is established, or it is withdrawn from use. From the tone of these two publications and remarks I have heard at psychiatric meetings, bcnzodiazepines are running the risk of being banned. Surely this must not happen. If we are concerned about our image and our efficacy in dealing with a whole range of psychi atric problems, the benzodiazepines must be retained as part of our armamentarium. On reflection, the College statement on benzodia zepines and dependency4 leaves one with an uneasy feeling. One does not get an impression that its authors and the participants at the special meeting on 10 June, 1987 were individuals who spend the major part of their time at the 'coal face' dealing with patients. This group seemed to consist mainly of psy chiatrists with a major commitment to academic work or with a bias in their work towards under taking trials with new drugs. It would seem to me that individuals with a major commitment to academic work or to the evaluation of new drugs are not well placed to form accurate or impartial views about the place of benzodiazepines in contemporary psychiatric practice. The menially ill on remand in prison DKARSIRS I am extremely concerned about the fate of the men tally ill on remand in prison. When I took over as senior registrar at the start of the year, I understood that my duties included assessment of people on remand in Brixton prison and that often this would lead to admission to hospital. This system seems to have broken down completely. Virtually all the prisoners end up in police custody. Two recent referrals underlined this point. Both went to court two days after the referral and both went to police custody. Thus neither is available for assessment. In the first six months of this year 10 out of 12 referrals went to police custody within 14 days of referral and were not seen. I was able to sec one man only because the prison medical officer 'pretended' he was unfit to go to Court. I would be interested to know whether others have similar experiences or any suggestions as to what we might do. If these occurrences are widespread, they can only lead to further delays within an already overburdened prison system. Meanwhile patients with mental illness who require hospital treatment are not receiving it.
N. J. MARGERISON St Luke 's Woodside Hospital Woodside A venue Muswell Hill, London N10 The proposed Community Treatment Order DEARSIRS It is exasperating when all one's charm, wiles, strata gems and threats fail to persuade a symptomcontrolled patient with chronic psychosis that his/her continued wellbcing depends on continuing treat ment. Nevertheless, I am extremely grateful to Lucy Scott-Moncrieff for enumerating all the excellent clinical and practical arguments, as they concern the individual patient, for us. I was sorry also to read that she shared my fear that this development could give further encouragement to the development of an even more threadbare community-based service. In view of the profession of the author, I would like also to have read her views on the legal implications of this proposal, which seem to me to arise from the basic step of taking away certain civil rights from an already highly under-privileged section of the com munity. Are there not also political issues? If a future Government decided that the Community Treat ment Order was a useful form of control of political dissidents or even Stonehenge-loving hippies, would we come out of the affair any more nobly than our Russian colleagues? https://doi.org/10.1192/pb.12.10.453 Published online by Cambridge University Press
|
10.7146/nja.v22i40-41.5202
|
cc-by
| null | null |
openalex
|
Light and The aesthetics of the perception ABSTRACT Light seems to be a very changeable size in our build environment. Being an immaterial building stone, light takes a very liquid shape in our designvocabulary. It consists of an invisible material -photons -and therefore it takes no specific form in itself but is only articulated through the meeting with form. Therefore, since form has been the major theme for the aesthetics up until now, giving form to light is a complex and challenging task and reducing it to Lux and measurable numbers only an escape from facing what is actually percieved. In this way light seems to suffer from what can be called the dichotomy between the aesthetics of the objects and the aesthetics of the perception -as stated by Boehme. To improve practice this article conducts a study of our perception, focusing more on the effects of light and less on the physical light (Lux) . By doing so the article tries to give a better understanding of the differences of the regional lighting cultures and the influences creating the differences. The article tries to establish a link between the regional daylight and the use of artificial lighting, showing that daylight, as a background, along with our perception, are determinant factors for how the artificial lighting and the brightness of the room is percieved. The article hereby suggests that light is not an absolute factor. This means the end of the dichotomy between daylight and artificial light -often expressed by artificial light replacing daylight -instead this article tries to establish a dialogue between the daylight and the artificial lighting. The article describes how light -this intangible building block -can become a more workable size in the aesthetic and architectural practice of today. KEYWORDS Light, daylight, artificial light, aesthetics of the perception, architecture, wellbeing, human-ecology, experienced brightness, atmosphere, design. CARLO VOLF is a PhD student at Aarhus School of Architecture, working on a PhD thesis on the interaction between daylight and artificial light its influence on our general wellbeing in Scandinavia. His earlier publications include both practical and theoretical works. In Lightstories 1998 Lightstories -2001 he wrote different light chapters using artificial lighting, telling stories with light, working on developing a more sensual light. In 2006 In -2009 he researched and experimented using a healthier lighting in hospitals in a research project made at Rigshospitalet in Copenhagen. His publications include: Computer-and daylight controlled lighting at hospitals (Lys 2009) and Daylight atmosphere in the dark corridors. (Ugeskr. Laeger 2009) Aesthetics is a study of the interaction of our senses with our emotions, in shor, our taste. A study of our most important sense, the vision and the influence of the interaction between daylight and artificial light on our vision is of great importance to a deeper understanding of the aesthetics. This study will reveal two major factors of importance; on one hand the regional differences and on the other hand an insight in our
vision and the influence of our vision on what is being seen. Linking these two factors together, this article will give an answer to this fundamental question: Why do Scandinavian countries prefer a warmer artificial light while southern countries use a cooler lighting? A study of great importance to the aesthetics, showing that the aesthetics is not an absolute, global phenomenon, but more likely to be considered a regional phenomenon, Mona Lisa in Paris not being the same as Mona Lisa in Copenhagen. This paper argues against the existing dislocation of light and the separation of light from form in order to create a more human-ecological point of departure for the future architecture in Scandinavia.
Introduction: Lighting belongs to the new aesthetics as described by Gernot Boehme. Light is an everyday aesthetic that surrounds us and forms a background for our lives. Boehme says "I believe, bringing me to my fourth thesis, that architects and designers of our time can ́t learn a lot from the theory of aesthetics, on the other hand I believe that the aesthetic theory can learn a lot from the practice" 1 . This text will add a critique to his thesis. If this 4 th thesis should give meaning, practice needs to be untied from the leaches that tie it to a functionalistic regime. Since "Mechanization took command" 2 a lot of things have changed and functionalism has reduced light into measurable figures in the Western World in the attempt to produce and plan the best physical environment for human beings. But this has been a poor attempt, reducing experience and atmosphere to zero-value and neglecting the emotional wellbeing of the inhabitants in our build environment. That the aesthetic theory can learn a lot from the practice is therefore not a convincing thesis for the building practice of today in Denmark or for that sake anywhere else in the industrialized World. Working in practice with artificial lighting often mean adding quantities of light to a building instead of adding qualities of light to the atmosphere. I often feel that there is a missing link between the building and the light, to improve the aesthetic conditions light should be linked to the architecture -creating an architecture as a function of the light. That is an architecture where form articulates the light and the light relates to the room. I will not exclude the overall possibility that The Aesthetic theory can learn a lot from the practice, but dealing with light, the practice seems to be submitted a functionalist way of thinking where light is added in quanta giving the atmosphere a second rate status, leaving the atmosphere so to say "as a function of the function". From a functional point of view the vision is dependent on sufficient amounts of light to see, e.g. reading a book, welding a software part, etc. But this method of thinking deals with a dislocated environment, separated from its surrounding environments. In this sense -leaving all other factors out of the equation -this way of thinking reduces light to a measurable factor, but also a factor blind towards the actual experience -and therefore often misleading. This exclusion of the environment dates back to Adolf Loos ́ Ornament and Crime and his 30 years struggle against the eclectism. But as Chombart de Lauwe later writes:"By limiting the meaning of the word functionalism -giving the word a far too narrow and technical meaning -any individual freedom was suppressed as if it was all about providing a shelter for rabbits or mice rather than for humans. 3 " The fight against the ornament was primarily based on a consensus of fighting for a better world. If only the symbol of the evil -the ornament -was exterminated all evil would perish with it and a new international architecture would emerge an architecture which in itself would be good if only it denied using ornament. Off course this didn ́t happen. Instead we got clean, white rooms stripped of all reflecting and shaping surfaces, an empty holster to be filled with only light as decoration. But light without form is the same as form without light. So we need to relocate the light and continue this debate and question this development in order to bring us upon a more sustainable course where interplay between daylight and electrical light, between light and form is examined further. From a human-ecological point of departure, light is -only visible because of darkness. If everything is illuminated equally, then we might as well be blind or blinded. We find ourselves in generally surrounded by more and more artificial light. We see it today in the cities, where focus on "Dark Skies" attempts to limit the amount of artificial light we emit out into the space. This light is preventing us from seeing a starry night. We see it when we are driving cars in full daylight with the lights on. Apparently based on the ground that this is more secure, something that can ́t be justified from a professional lighting point of view, perhaps in reality rather challenged as car lights provide glare and disadvantage compared to other vulnerable road users? Have we come too far into our artificial and excited use of artificial light, so that we are incapable of even seeing it? Are we -quite literally -blinded by the light?
A Ground Zero: Often the dichotomy between daylight and artificial light relates to another dichotomy: Nature contra technology. Sigfried Giedion traces this dichotomy back to Rousseau ́s time. With the rising industrialization in the 18 th century the contradiction between nature and culture is established -and with culture being more and more based on technology -this contradiction today seems more rather to be one between nature and technology. Modern architecture developed after the 1. World war -as a result of social and political revolutions -and driven by technological and engineering developments of new cheap building materials such as steel, glass and machinery. This results in a new way of dealing with daylight. The Crystal Palace by Joseph Paxton at the Great Exhibition of 1851 in London is one of the earliest examples of a new architecture of glass and light. This creating a new aesthetic of openness, light and a healthy and clean physical environment. This healthy and democratic architecture has played a major role in the 20 th century architecture, bringing light into the buildings -and as we shall later see, forcing a new way of articulating the light -building deeper, with lower room-heights and using UVfiltered and colored glass to protect against overheating, accentuating the use of another kind of lightnamely the electrical light: Until the end of the 19 th century no efficient artificial lighting was available, the build environment relied upon the natural existing daylight and light from fire, petroleum, etc. In 1879 Thomas Alvin Edison patents the incandescent bulb and starts a network of power-suppliers for electricity worldwide. Making it possible to bring a soot-free, safe and healthy lightsource into the buildings. Developing fast, electrical lighting and illumination becomes one of the main attractions in the Fin de Siècle architecture. Artificial lighting plays a major role in the industrialization, creating an improved visual environment, so to say "prolonging the day". Perhaps electric lighting expands so quickly, because it is one of the founding stones of the industrialization. There is no doubt that it has an enormous impact on our productivity and on both our physical and psychological development and behavior -making it possible to build larger buildings and creating longer working hours. However this success of the electrical lighting has not only had beneficial effects, it has also drawbacks. One of these drawbacks is the neglect of the use of daylight -along with a neglect of nature itself. This may seem odd, daylight being considered a very precious factor in the build environment of Scandinavia, in fact in all modernist architecture. But it is none the less rather a rule than the exception that the light in modernistic buildings is being transported into the room without any articulation, reflection and optical efficiency. Let me elaborate on this: Light in itself is invisible to the eye, it is only reflected light that is visible to the eye -and therefore only the meeting of light and form that is determining the optical efficiency. Glass and electric lighting do not in itself provide an experience of brightness. Instead what we have is a sense of brightness and of transparency -based on the presence of so much glass -but actually it provides reduced optical conditions for the eye: As someone once said to me: "During the day I shield the daylight, daylight annoys me, especially when I look direct at the windows" Glare from the windows cause a feeling of a dim and gloomy lighting in the daytime, all focus seem to be outside the building, not inside. Much of the present architecture today is like this, squeezed and concentrated -often located on expensive square meterswith many floors. But of what help are all the glass facades when a low room height or a deep-plan building is reducing the natural skylight and the amount of daylight in the room? Walking in to an average deep-plan office, you have curtain-wall glass on the façade providing a large view, reducing the feeling of a border between indoor-outdoor. However well planned and beautiful articulated this curtain-wall glass provides only little reflection and articulation of the daylight inside the building. Look at the reflecting surfaces adjacent to the glass: Carpets often dark for cleaning purpose, no details around the windows no windowsills. Actually the sills are often dark so that you focus on the view outside -framed by the window. Was it not for the electrical light, it would be an unpleasant experience working in this room, contrast between daylight and darkness being too large, seeing only silhouettes of persons in the room, feeling a gloomy atmosphere. In this way the electrical lighting causes a negligence of daylight. The electric lighting being the excuse for building deeper room-depths with lower room-heights and lower daylight factors. Leaving the electric light on all day. However it was not always like this, ventilation along with electrical lights -and medicine -has been the driving power for reducing this room-height to the minimum of 2.5 m today. With modernist architecture engaging with technology since the 1920-30 both ventilation and electrical lighting became a common mechanization of the building, replacing the earlier tall -but expensive -3.0 m ceiling heights, and reducing the window-height. The window loosing the original meaning of the word: wind and ow -wind and eye -fresh air and light. From 1930 -1950 almost 95% of all electricity in Denmark was used for lighting 4 -actually a bill for the electricity was called a light-bill. At this time there was a limited access to electricity and electricity was quite expensive. From the 50s and up the power-supply all in all were improved and the electricity costs were stepwise reduced. Hence a big leap is observed from the early ́60s where both the use of electricity and the available electric equipment doubles within a short period of 5 years. The following reductions in electricity costs only accelerated the use of more electricity in this period, and what is very symptomatic for this change is the exceeding use of artificial lighting in the buildings. In Denmark a final step further away from nature and natural daylight was taken in 1977 building legislation (BR77). Due to the energy crisis the window area is reduced to 15% of the wall-areal. This meant that heating energy was saved, but more electrical lighting was required. In reality making the people more dependent of the electrical lighting and less dependent of the daylight, thereby creating a new building-culture in Denmark -this being a Ground Zero for the lighting in Denmark. So, all in all light seem to be a very changeable size.
How to move on from a Ground Zero? Why do we need to bring the light out of this shadow of functionalism? Because we are forced to do so. We are in a situation where we need to reduce our consumption of artificial lighting and this being said we need to use the existing daylight. In short we need to spend the light more intelligently and to look more closely at the interaction between daylight and artificial light. Not only for the sake of the environment but also for the sake of our general wellbeing when we keep in mind that we spend most of our time -up to 90% of the time 5 -in the indoor environment. To improve the chances for practice to create a better environment, let us in the following have a closer study of two factors of importance; 1) the vision and 2) the use of regional lighting conditions. 1) The vision always adjusts to a given light-level, only being able to perceive momentary differences in brightness. At a given time the eye can approximately distinguish up to 20 different lighting intensities. This makes seeing a relative sense -not an absolute sense -and a momentary sense due to the inability of the eye to remember absolute brightness and color. That is why light is a changeable size. In the 1930s in Denmark we had a standard saying that 30 Lux on the desk was sufficient for reading, today in Denmark we have 400 Lux and in Sweden 500 Lux. In future engineers plan for even more Lux -I recently heard 1.200 Lux! This is of course a result of technical development but increasing the level to 1.200 Lux will not solve a problem of our perception of brightness nor of darkness. Light being a relative factor. The mere use of more light will therefore not result in a better physical environment. Let me flesh this out in an example: In Aarhus I have passed through a certain passage hundreds of times over the years and found the existing electrical lighting in these passages, being very sufficient and very well designed. But then, this autumn, when I returned an evening, a new passage-way had been build and added to the existing one prolonging it through to another building complex. The new lighting in this new corridor all of a sudden made the existing passage look gloomy and insufficiently lit, appearing completely different. Though it had not changed at all! This explains the following question: Why does our physical lighting environment change from 30 Lux to 1.000 Lux? The answer to this question is that we don ́t observe this change. As the earlier city major of Copenhagen Ritt Bjerregaard, recently said at Earth Hour 2009: We get used to the light -the more light we use the more light we need! Now to argue to what extend a recent amount of light (Lux) is, is a very interesting and complex matter. But this observation means for sure that there seem to be no absolute figures, no foundation for absolutism in terms of lighting! The human vision is a quite complex matter. Also when it comes to distinguish color. Electrical light appears warmer on a background of daylight, daylight influencing both the experience of brightness and color. The electrical light is being like "an artificial sunbeam" striking the room and creating bright and warm colors on the faces we see. Therefore we use artificial light as a way of providing artificial sunshine in our environment. We do not use artificial light only as a mean of getting more light but also of getting a warmer light! To explain this further we need to talk about the Purkinje-effect or the relationship between the photopic day vision and the scotopic night vision. This effect describes that the relative sensations for various colors alter as the brightness values are reduced to lower intensities. The Purkinje effect operates only within the eye, creating this change of color by changing from day vision to night vision. For example a reddish purple will be seen to vary from reddish purple to a more blue violet. Sensitivity to color changes along with the intensity of the light, from a yellow-green peak-sensitivity (555 nm) and to a more blue-green peak-sensitivity (507 nm) this means that we see e.g. warm colors in another way at night -as more "white". This could be the answer to the initial question: Why do we in Scandinavia tend to like a warm electrical light compared to the cool daylight? New lighting technologies like LED and CFL make it possible to change to any color you would ever like, breaking the existing restricting technology of the incandescent bulb with its characteristic warm color (K 2700). Yet we don ́t observe any change in preferenceactually the new technology is only miming the old incandescent light color. So why this warm color? Because it evokes a feeling of an electric "sunbeam"? As human beings we have developed a unique color vision. Color vision defined as: The capability of discriminating lights (scattered light as well as light sources) on the basis of the spectral content of the light, even when those lights are of equal subjective brightness. Now picture the rainbow: What you see appearing to have discrete bands of color is not a physical phenomenon, it is not out there arisen from the radiation in the sky. If you measure the radiation with a spectrophotometer, you will find that the wavelength of maximum intensity as a function of the radial distance across the rainbow decreases smoothly and monotonically from the outside to the inside of the bow. The apparent discreteness is an artifact of our photo-pigments (chromophore and opsin) and the neural processing of our photoreceptors' output to our brain! The brain is creating what we see, actually what we see is not what is actually there, but what the brain interprets from the incoming signals in the eyes. The specifics of our unique color vision are still somewhat mysterious. One thing however is clear: The best known predictor of what sort of pigments will be expressed by any given animal, is the pigments expressed by its nearest living relatives. In our case this means the color red because of the color of our skin -not quite accidentally also bearing a lot of synaesthetics, or as by Goethe a Sinnlich-Sittlich 6 effect, like warm, near, erotic. To an evolutionary biologist this makes a lot of sense because we as primates developed in a our own way in the Mesozoic age, from small rodent-like mammals that were most probably nocturnal to the evolution of a unique vision -our tri-receptor color-vision. In this evolution from mammals, segments of color vision were lost, only to be regained for some species of primates, by gene-duplication. Therefore other mammals like for example dogs and cats generally only have two-receptor color perception systems, which can distinguish blue and green-but not the reds. In other words we use three types of chemical photoreceptors with red as a new color. This adaptation to see reds is particularly driven by its importance for our survival, since it leads to identification of potential mating partners. This is one good reason why we love the warm color of light -it affects our limbic system and our deepest emotional instincts because it supports our very survival and wellbeing. It is important to note that it is not as much the light itself, but more its effect on the faces we meet or the inventory we see, etc. If we combine 1) The fact that our vision actually only can see the warm colors on a background of daylight with 2) The fact that we are evolved as creatures of light and even more precisely as creatures of sunshine, we derive at an explanation of why the southern culture of light is different from ours: Simply because they switch from the day vision to the night vision in a relative short period of time due to their regional environment. When adapted to the night vision the yellow light appears all of a sudden white because of the light-adaption of the eye, this goes for any color of white -cold or warm -since any color in the darkness seem "white". Southern countries are therefore not as dependent of the warm color of light than we tend to be in Scandinavia, because they don ́t have the same long transitions between darkness and light, no background of daylight when they use the artificial light. This is an explanation for why you do not find the warm light when you look at countries nearer to the Equator. 2) The use of regional lighting conditions; Denmark and Scandinavia have very long transitions between the day and the evening. While near the equator the diurnal daylight is followed by the nocturnal darkness in an extremely short period of only 15 minutes all year round; Denmark has a dusk period of approximately 4 -7 hours of the total 24 hours cycle, with large variations during the year. This being the result of our latitude at 55 th N in Denmark, meaning that the sun has to travel a smaller distance compared to near the equator in the same span of time, e.g. 1 hour at the equator equals 1/24 of 40.000 km, while 1 hour at 55 th latitude only equals approximately 1/24 of 31.500 km. This, together with the decline of the axis of rotation by 23.5 degrees, explains the differences in the regional rhythms of daylight. Duration of the twilight period between dawn and sunrise varies greatly depending on the observer's latitude -from a few minutes to several hours. At the North Pole there is little or no difference between day and night, while a big difference is observed between summer and winter. Moving to the equator reverses this fact: Little if no difference is observed between summer and winter, while a big difference is observed from day to night. In Denmark and Scandinavia this means that electrical lighting during a large part of the day is used on a background of daylight. This interaction between daylight and electric light has a great impact on the aesthetics. In practice changing both our perception of light and of color. In Scandinavia we can neither neglect the use of artificial light nor neglect the importance of sufficient daylight. We cannot just pour artificial light in the building where there is not enough daylight and we can ́t base the lighting on daylight alone. Alvar Aalto is a good example of this pragmatic relationship between daylight and electrical light, recognizing the beauty and strength of artificial light though never neglecting the vital daylight, Aalto used the daylight in his buildings together with the artificial light. He realized the fact that in Scandinavia we can ́t live with only daylight nor with only artificial light. Aalto was one of the first to break with the modernist thoughts of creating an international style, turning away from a pure rational and functional architecture. Working with the local experience and emotions as functions equally important as the more physical functions, Aalto combined daylight and artificial light, working with light-zones, integrating artificial light with the architecture, locating the light in the room, and so forth. He understood that light and wellbeing is a result of daylight and artificial light working together, not just replacing each other, but reinforcing each other. Aalto designed several lighting fixtures for his buildings working with the limited amount of regional daylight as an inspiration rather than a limitation.
Conclusion: The functional approach towards light has not always been the norm -actually it is rather a recent exception to a human-ecological approach -and is derived out of the post First World War ́s implement of technology in our build environment. With electrical lighting dating only 130 years back, it ́s actual influence on our society and on our way of living and thinking is however gigantic and incomprehensible. Before this revolution of the light, the sufficient amount of light was measured in candles (candelas) and the perception of brightness was therefore not only a result of the amounts of light, but was dependent on architectural forms and ornaments on doorways and staircases, for example. One could say that this was a culture where light and form where closely connected. For example, in the "lysedug", a white tablecloth reflects a single candlelight out into the entire room, or the ornamented steps of a stairway lighting up the darkened steps, reducing the possibilities of falling accidents. This location of the light in the build environment shall be seen in contrast to the dislocation of the light mentioned earlier, a dislocation caused by/causing the standardization of the light in the 1960 ́s, reducing light into measurable quantities not qualitites. In the same perspective the subsequent method of dealing with Lux shall be seen in contrast to dealing with candela/sqm and the perceived brightness of the surfaces in the room. From an ecological point of view, we need to reduce our energy consumption -in Denmark by 4 percent -which is a new challenge. Ever since we started using electricity, we have only used more and more energy each year. Lighting takes up to 20 percent of our total use of electricity, to challenge our way of using the light is in this sense quite necessary if we look into the future. But how do we do this without reducing the wellbeing of the occupants of the build environment? Much of the explanation to this question can be found in this essay, as described there seem to be a tendency towards using more and more light in our culture. Taken into consideration that the eye can only see up to 20 different lighting intensities at a time, it is possible to construct a daylight-artificial light environment where these 20 levels are not exceeded, meaning that the artificial light responses to a given daylight at any time. Daylight being the background determinant/chord for the experienced brightness of the artificial light, means that they both have to be taken into consideration. Again this explanation speaks against any dislocation of the light. Light being related to the daylight of the region and located in a room -not dislocated. This means that we can abandon any absolute tyranny of Lux and instead work with a more relative, perceived brightness -in this way we can reduce the amount of electrical light in the evening and use artificial light together with form to avoid exceeding the maximum capacity of light in the eye, when there is daylight. In this way we can regain the darkness at night and yet be able to see and inhabit the large scale buildings and low roof /deep floor offices that seem to be an inevitable result of our modern urban society. In this case, the amount of light is not important in its physical form (Lux) but in its "perceived form" (candela/sqm). Before "the revolution of light", a candle could easily light up these passageways because they were shaped to our perception. But today even the exceeding use of artificial light can ́t prevent elderly people from tripping on a staircase with no perceptual form (top: Stairway and bottom: Doorway) Fig. 3 : To locate the light in a room (the brightness) a simple "Scale of light" is introduced. The luminance is not measured in absolutes but in relative significant-areas of luminance relating to each other (in 9 visual levels). The scale tells about the character of the room, how the distribution of light is and where it can be improved. In the casestudy shown here the apsis of the church appears too dark in relation to the surrounding space. (Red lines indicate it is 6 levels darker and that it changes abruptly from 400 cd/m2 to 5 cd/m2) Overall point of view: Our build environment is at its best a reflection of these conditions: location, orientation and activity. Light is one of the important factors revealing space, time, activity and focus. Locating the light is of great importance for this to happen. Our parts of the world was former known as places ihibitabilis, this meaning they were not able to house human beings! Electrical lighting has along with other new technologies made this possible, this mechanization, the lack of sunlight and use of electrical light has created a special culture and architecture in the Northern countries -an interaction between daylight and artificial light. In the future we need to focus on a human-ecological lighting and study how light affects us and how we perceive the light, as humans located in a region. All in all -to relate this to Boehme -this conclusion shows that light is not only being out there in physical form or Lux. Light is also a relationship between object/region and subject. In other words, light belongs to the atmosphere. 1 Annual Conference of the Nordic Society for Aesthetics 2010
Fig. 1 1 Fig.1 and 2: Examples of form and light working together, the light being located in the room. In this case, the amount of light is not important in its physical form (Lux) but in its "perceived form" (candela/sqm). Before "the revolution of light", a candle could easily light up these passageways because they were shaped to our perception. But today even the exceeding use of artificial light can ́t prevent elderly people from tripping on a staircase with no perceptual form(top: Stairway and bottom: Doorway)
Boehme, Atmosphäre: Essays zur neuen Ästhetik (Frankfurt am Main: Suhrkamp, 1995), 17b 2. Sigfried Giedion, Mechanization Takes Command: A Contribution to Anonymous History (Oxford: Oxford University Press, 1948) 3. Paul-Henry Chombart de Lauwe, "Soziologie des Wohnens," Bauen und Wohnen, Vol. 16, No 6, 1961 4. See the study "Boligers elforbrugsfordeling på slutanvendelsesgrupper 1946 -2030" carried out by ELFORSK 5. According to research, this is the case in Denmark
Atmosphäre, G. Boehme, 1995 (p.17b.)
Sigfried Giedion, 1948
Soziologie des Wohnens, Bauen und Wohnen 16.Årgang, 1961, p.139, p. 218
Source: ELFORSK, "Boligers elforbrugsfordeling på slutanvendelsesgrupper1946 -2030"
In Denmark (SBI)
J.W.Goethes Zur Farbelehre, 1810
|
10.1186/s13756-019-0522-6
|
cc-by
| null | null |
openalex
|
Background: Increasing bacterial resistance to antibiotics is a serious problem worldwide. We sought to record the acquisition of antibiotic-resistant Escherichia coli (E. coli) in healthy infants in Northern Thailand and investigated potential determinants. Methods: Stool samples from 142 infants after birth, at ages 2wk, 2mo, 4 to 6mo, and 1y, and parent stool samples were screened for E. coli resistance to tetracycline, ampicillin, co-trimoxazole, and cefazoline by culture, and isolates were further investigated for multiresistance by disc diffusion method. Pulsed-field gel electrophoresis was performed to identify persistent and transmitted strains. Genetic comparison of resistant and transmitted strains was done by multilocus sequence typing (MLST) and strains were further investigated for extra-and intra-intestinal virulence factors by multiplex PCR. Results: Forty-seven (33%) neonatal meconium samples contained resistant E. coli. Prevalence increased continuously: After 1y, resistance proportion (tetracycline 80%, ampicillin 72%, co-trimoxazole 66%, cefazoline 35%) almost matched those in parents. In 8 infants (6%), identical E. coli strains were found in at least 3 sampling time points (suggesting persistence). Transmission of resistant E. coli from parents to child was observed in only 8 families. MLST showed high diversity. We could not identify any virulence genes or factors associated with persistence, or transmission of resistant E. coli. Full-term, vaginal birth and birth in rural hospital were identified as risk factors for early childhood colonization with resistant E. coli. Conclusion: One third of healthy Thai neonates harboured antibiotic-resistant E. coli in meconium. The proportion of resistant E. coli increased during the first year of life almost reaching the value in adults. We hypothesize that enhancement of infection control measures and cautious use of antibiotics may help to control further increase of resistance.Background Escherichia coli (E. coli) colonize the gut of newborns within hours after birth when the newborn is exposed to the maternal vaginal and fecal flora and to environmental bacteria [1, 2] . Under physiological circumstances E. coli living in the colonic mucus and its human host are symbionts . Nevertheless, virulent E. coli strains can severely sicken neonates and infants, causing enterocolitis, urinary tract infections, meningitis, and septicemia . Increased antibiotic resistance in E. coli has been observed in recent years ; antibiotic-resistant pathogens are generally a growing menace , especially in low-income countries, where prevalence of resistant bacteria is higher than in high-income countries . In Thailand, rates of antibiotic resistance in E. coli are high in healthy adults, food, food animals, and the environment . In literature, there are only few data on the prevalence of antibiotic-resistant bacteria in healthy newborns [19, 20] . Smit and coworkers have found a high rate of neonates (46%, n = 17) to be colonized with multidrug-resistant Klebsiella pneumoniae within 24 h of admission to a neonatal unit in Cambodia and they have claimed a high within-host diversity . It is well known that antibiotic exposure triggers the development of resistant bacteria . However, it is still a matter of debate how infants acquire resistant bacteria including commensal bacteria of the intestinal microbiota without any antibiotic selective pressure. We sought to investigate the acquisition of antibiotic resistance in E. coli from stool of healthy infants in Northern Thailand during the first year of life and to assess whether resistant E. coli persist in infant gut as well as whether these microorganisms are transmitted from parent to child. We also investigated which host and environmental factors and which bacterial virulence factors had contributed to frequency and duration of infant gut colonization by antibiotic-resistant E. coli.
Methods This cohort study was approved, according to the declaration of Helsinki 2000, by the ethics committees of the Medical University of Innsbruck (Ethics approval number: 260/2010) and the Faculty of Medicine, Chiang Mai University (Ethics approval number: 260/2533). Samples were collected between September 2010 and December 2012 at the Department of Pediatrics, Chiang Mai University, and the Meledek (Mother and Child) Hospital, both Chiang Mai, and at the Sanpatong District Hospital, all in Northern Thailand after informed consent of the parents was obtained. Characterizations of the resistant strains were completed in 2017.
Study participants All healthy singleton newborns of the participating hospitals that were born during the enrollment period were consecutively invited to take part in the present study. A healthy infant was defined as a newborn with good fetal adaptation (Apgar score at least 9 after 10 min; umbilical cord vein pH > 7.20) without any signs of infection (body temperature < 38.0 C; C-reactive protein < 0.7 mg/ dl or C-reactive protein not measured due to the lack of clinical signs of infection). Infants with syndromic disorders or malformations influencing the function of the gastrointestinal tract as well as immunodeficiencies, problems of enteral feeding for > 3 days or hospital stays > 14 days were excluded. Infants born to parents with immunodeficiency or malignancy or of mothers given antibiotics during labor were also not enrolled -antibiotic use in infants during the first year of life was no drop out criterion. The study protocol aimed collection of meconium samples during the first 48 h after birth, infant stool samples at ages 2wk, 2mo, 4 to 6mo, and 1y, and parent stool samples at infant birth. Families in which more than one infant stool sample was missing at the end of the follow-up were withdrawn.
Investigation of host and environmental factors associated with the development of antibiotic resistance Demographic and clinical data (gender, birth mode, complications, underlying diseases, antibiotic use, hospital, etc.) and environmental data (siblings, pets, infant feeding) were recorded by caregiving nurses or physicians through personal obtained questionnaires (Table 1 , Additional file 1).
E. coli collection and characterization Stool samples were collected by nurses and parents. Parents were trained during hospital stay in collecting a hazelnut-sized portion of freshly voided feces placed in a sterile sample container in a plastic bag, which was kept refrigerated at 3 C until transport to the laboratory. If culture within 24 h was not practicable, samples were frozen at -20 C until further processing (applicable for < 5% of all samples). Stool samples were plated on a MacConkey agar plate (Becton Dickinson, Cockeysville, MD) and another 4 MacConkey agar plates supplemented with defined concentrations of tetracycline (TET; 4 μg/ml), ampicillin (AM; 8 μg/ml), co-trimoxazole (SXT; 8 μg/ml), or cefazoline (CEF; 8 μg/ml) as described previously . As detection of E. coli from stool culture on MacConkey agar plates may suffer from poor sensitivity due to overgrowth, we have decided to include MacConkey agar plates supplemented with antibiotics. We have chosen TET, AM, SXT, CEF as screening antibiotics for two reasons: First, these antibiotics are widely used in human and veterinary medicine and especially in treatment of early childhood infection, thus resistance to these antibiotics are an important issue. Second, the use of tetracyline is obsolete in children below the age of 8 years due to severe side effects. Thus, detection of resistance in infants reveals interesting insights into the problem of creating a resistance pool due to wide use and its persistence. After growth on agar plates, differently appearing colonies from each agar plate were collected for further analyses. To confirm the hypothesis that colonies with same morphological appearance originate from the same clone, we have picked two identical appearing colonies from each plate. Furthermore, colonies with the same morphological appearance on different agar plates were frozen as well. All isolates were transferred to the Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, where the following analyses were performed. The procedure described above was based on a pilot study performed in 10 families in which 5 similar-appearing colonies from stool samples were genetically indistinguishable by pulsed-field gel electrophoresis (PFGE; see below). For identification, isolates were streaked on ChromID CPS medium (bioMérieux, Marcy-l'Étoile, France) and incubated for 24 h at 37 C. All red or white colonies were considered E. coli. Species identification was confirmed by Matrix Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) (Bruker Daltonik, Bremen, Germany). All E. coli strains isolated according to the protocol detailed above were tested by disc diffusion for resistance to 16 different antibiotics (AM, ampicillin; AMC, amoxicillin-clavulanic acid; CIP, ciprofloxacin; CN, cefalexin; CPD, cefpodoxime; CRO, ceftriaxone; CXM, cefuroxime; ETP, ertapenem; FM, nitrofurantoin; FOS, fosfomycin; GM, gentamcin; MEC, mecillinam; SXT, co-trimoxazole; TET, tetracycline; TMP, trimethoprim; TZP, piperacillin-tazobactam) on Müller-Hinton agar plates according to EUCAST clinical breakpoints (V06/ 2016) or, for TET, using CLSI standards .
Genomic characterization by PFGE PFGE was performed only in E. coli strains with the same resistance pattern which were isolated in at least 3 different sample time points (infants) or in at least one parental stool sample and two infant samples (resource limitations precluded testing of all E. coli cultured from stool samples). PFGE was performed according to a standardized protocol for molecular subtyping of E. coli (PulseNet, CDC). PFGE profiles generated by XbaI digestion of chromosomal DNA were analyzed using BioNumerics 3.5 software (Applied Maths, Sint-Martens-Latem, Belgium) and clusters were defined as described .
Persistence and transmission E. coli strains with the same antibiotic resistance profile and PFGE cluster isolated from at least 3 different samples from one infant were defined as "persistent". In families, E. coli strains with the same antibiotic resistance profile and PFGE cluster isolated from at least one parental stool sample and at least 2 different infant samples, "transmission" of the strain from parent to infant was inferred.
Multilocus sequence typing (MLST) and phylogenetic grouping All persistent and transmitted strains underwent MLST, as did a control group of 7 strains only found once. Gene amplification and sequencing were performed using primers specified at the E. coli MLST web site (http//mlst.warwick.ac.uk/mlst/dbs/Ecoli). Sequences were analyzed using the software package Ridom SeqSphere 0.9.19; sequence types (ST) were computed automatically . Phylogenetic groups of the strains were determined using Structure 2.3.X software based on the concatenated sequences of the 7 housekeeping genes used for MLST (https://web.stanford.edu/group/ pritchardlab/structure.html). MLST was performed at the Institute of Hygiene of the University Hospital Münster.
Virulence and adhesion gene typing Using multiplex and single PCR, all persistent and transmitted E.coli strains as well as the 7 strains in our control group were investigated as described for genes linked with extra-and intra-intestinal pathogenicity, including those encoding for adhesins, iron acquisition, protectins, toxins or invasins, and other virulence factors (Additional file 1).
Statistics A sample size of 150 study participants was calculated to estimate the prevalence with an accuracy of ±8%. We applied the Fisher's exact statistical test to analyze our contingency tables, valid for small sample sizes. A p-value of < 0.05 was considered as statistically significant. Univariate analysis of variables of interest was applied. Associations were calculated by using Epi. Info 2.2.2.2 software (CDC, Atlanta) and presented as prevalence ratios (PR) including 95% confidence intervals (95%CI).
Results During the enrollment period 437 babies were born in the participating hospitals (Fig. 1 ). Among the latter, 169 newborns and their parents fulfilled the inclusion criteria and consented to participate in the study. At the first sampling time point (within 48 h after birth) 169 meconium, 169 maternal and 164 paternal stool samples were collected. During the whole study period 27 families were culled due to lack of more than one stool sample, thus 142 families completed the study (Fig. 1 ). Among these 142 participants stool samples from 4 infants were not received at 2wk and from other 8 infants at 4 to 6mo. Ninety of the 142 infants were born at Chiang Mai University Hospital (63,4%), 31 at Chiang Mai Meledek Hospital (urban child birth clinic) (21,8%), and 21 at the rural Sanpatong District Hospital (14,8%); 75 were male (52.8%) and 67 female (47.2%). All infants were born between week 32 and week 42 of gestation, with 20 (14.1%) before week 37 and 122 (85.9%) were term births. In 103 cases vaginal birth was spontaneous and in 7 it required vacuum support; 32 babies were born by cesarean section (Table 1 ).
Prevalence of resistant E. coli in parents One-hundred forty stool samples from fathers and 142 from mothers were included in the study (two fathers were not parental caregivers). In 9 (6%) paternal and 7 (5%) maternal specimens E. coli showed no resistance to any of the 4 initially tested antibiotics. Prevalence of carriage of resistant E. coli were lowest for CEF (37% fathers, 29% mothers), followed by SXT (70% fathers, 62% mothers) and AM (84% fathers, 86% mothers, Fig. 2 ), and highest for TET (87% fathers, 87% mothers). Prevalence of resistant E. coli in meconium and development of resistance during the first year of life Forty-seven (33%) of the 142 tested meconium samples yielded E. coli and all of them showed resistance to at least one of the four tested antibiotics (Table 1 ). Prevalence of carriage of TET-resistant E. coli was 30% (n = 42), AM-resistant E. coli were found in 25% (n = 36), SXT-resistant in 20% (n = 29), and CEF-resistant in 8% (n = 12). Prevalence of carriage of resistant E. coli steadily increased during the first year of life. For TET, resistance proportions were 39% at 2wk, 46% at 2mo, 65% at 4 to 6mo, and 80% at 1y. The latter almost matched parental resistance proportions. Tendencies were similar for AM, SXT, and CEF (Fig. 2 , Table 2 ).
Antibiotic resistance profile of isolated E. coli strains All resistant E. coli strains (n = 1223, 558 parental and 665 infant) were tested for multiresistance against 13 additional antibiotics (Fig. 3 ). The most common resistance pattern in samples was quadruple, viz., resistance to 4 antibiotics (24% in parents, Fig. 3a ; 27% in infants, Fig. 3b ); most such samples (84%) were resistant to SXT, AM, trimethoprim (TMP), and TET. In 179 strains (86 parental and 93 infantile) single resistance was found, resistance to TET was most prevalent (67%). Multiresistance to 11 different antibiotics was found in 10 samples (1%). None of the tested strains showed resistance to ertapenem. Distribution among children and parents was similar. Third-generation cephalosporine (ceftriaxone) resistant E.coli were found in 4% of meconium samples, 7% at 2wk, 8% at 2mo, 10% 4 to 6mo and 18% at 1y. Prevalence of third-generation cephalosporine resistance after 1y almost reached the prevalence in adult samples (20%) (Table 2 ).
Frequency of persistent E. coli strains in infants and frequency of transmission of resistant E. coli from parents to infants In 39 families criteria for further PFGE investigation were met (see methods). In 8, the same PFGE clusters were found in parents as well as at least twice in infants ("transmission"); persistence was found in 8 infants. In 4 of the 8 persistent (50%), the same strain was found in one parent (transmission and persistence). Paternal transmission was found in 3 families, maternal transmission in 5. In most (n = 9) of the families meeting criteria for transmission or persistence E. coli showed quadruple resistance to SXT, TMP, AM, and TET (75%). Persistence from meconium sample to half-year stool sample was found in 3 infants, and once each from 48 h In 2 infants identical strains were found in meconium sample and in stool samples at 2mo and 4mo but not in 2wk stool sample.
Association of persistent and transmitted E. coli and virulence factors Molecular examination for various virulence and adhesion factors of E. coli strains with PFGE-confirmed persistence or transmission revealed similar distributions of these factors in both transmitted or persistent E. coli and non-transmitted and non-persistent E. coli (Additional file 1).
High diversity of E. coli strains by MLST MLST revealed a high variety of E. coli sequence types among both persistent or transmitted isolates and the control group (Fig. 4 ). ST10 (phylogenetic group A) was identified 3 times (all persistent or transmitted strains), ST69 and ST95 twice, and ST156, ST38, and ST538 once each. The 9 remaining E. coli isolates could not be assigned to any sequence types. Strains with identical STs were found in only 3 families (Fig. 4 ). Equal numbers of persistent and transmitted E. coli strains belonged to phylogroups A, B2, and D (n = 4 each). A similar distribution was found in the control group of transient strains.
Influence of host and environmental factors associated with the development of antibiotic resistance The difference in prevalence of resistant E. coli in infants who received antibiotic therapy during their first year of life and in infants without any antibiotic therapy was assessed using the questionnaire. Altogether 88 infants (62%) did not receive any antibiotics during their first year of life, whereas 54 infants needed antibiotic treatment (Table 1 ). Antibiotic therapy did not affect the prevalence of resistance. The distribution of resistance patterns was similar in both groups and at the age 1 year of life all infants in our study harbored at least one resistant strain (resistant against at least one of the four antibiotics used for screening) in their gut. Early prevalence of resistant E. coli in meconium was lower in children born by caesarian section (PR: 0.75; CI: 0.60-0.94, p 0.016) compared to vaginally born children. Preterm babies also showed less resistant E. coli compared to children born >=37wk of gestation (PR: 0.73; CI: 0.58-0.92, p 0.023). Having been born in the rural hospital was associated with a higher prevalence of resistance (PR: 1.84; CI: 1.06-3.22; p 0.003). Antibiotic usage by the mother during pregnancy showed to be protective (PR: 0.76: CI: 0.61-0.97; p 0.028) (Table 3 ). None of the investigated determinants, e.g., birth mode, kind of feeding, exposure to pets, and primary care was associated with persistence or transmission of resistant E. coli.
Discussion We investigated the colonization of antibiotic-resistant E. coli in stool samples of healthy infants in a birth cohort (142 families) over a one-year period. A culture-based approach was chosen due to several advantages compared to other methods like sequencebased tools especially the possibility to characterize single bacterial species in a stool sample. This method is unique for a big sample size as in the present study and a long observation period of 1 year, however, it might have limitations which are discussed below. In particular we found high proportions of resistance to TET, AM, and SXT, ranging from 20 to 30%, in E. coli from meconium of neonates in northern Thailand. The prevalence of antibiotic-resistant E. coli increased during the first year of life, reaching almost the same proportions as in parental samples, with > 70% resistance to TET and AM and 66% to SXT. Resistance against CEF was less frequent, with 8% in meconium samples and 33% in parental stool samples. In 8 children (6%) resistant E. coli strains persisted over at least three sampling time points. Early transmission of resistant E. coli from parents to children was identified in 8 families (6%). In neither persistent nor transmitted strains an association with any tested virulence and adhesion factor was observed. The neonatal phase is crucial for intestinal colonization with E. coli [1, 28, 29] , although this may occur even during the fetal period . Of our 142 meconium samples, 47 yielded resistant E. coli (33%). Compared with other studies this rate of A B Fig. 3 Overview of single-resistance (R1) or multi-resistance (R2-R11) patterns of E. coli strains in parents (a) and infants (b). The most prevalent resistance pattern is illustrated in black, the next most in light grey, and others in dark grey. (tested antibiotics: AM, ampicillin; AMC, amoxicillin-clavulanic acid; CIP, ciprofloxacin; CN, cefalexin; CPD, cefpodoxime; CRO, ceftriaxone; CXM, cefuroxime; ETP, ertapenem; FM, nitrofurantoin; FOS, fosfomycin; GM, gentamcin; MEC, mecillinam; SXT, co-trimoxazole; TET, tetracycline; TMP, trimethoprim; TZP, piperacillin-tazobactam) antibiotic resistance is high: In Innsbruck, Austria, among 21 neonates studied for resistant E. coli during the first 48 h of life, resistant E. coli (resistant to TET, AM, and SXT) was observed in only one stool sample (4.8%) . AM-resistant E. coli were found in 27 of 824 (3.3%) neonatal stool samples in Lithuania . The differences in resistance prevalence may be inferred from differences in study design and geographical background depended on the study location. Countries belonging to the Organization for Economic Co-operation and Development (OECD) are generally wealthy. In asymptomatic infants antibiotic resistance is much higher in non-OECD countries than in OECD countries: The pooled resistance prevalence in OECD countries to TET was 37.7% (25.9 to 49.7%), to AM, 37.6% (24.9 to 54.3%), and to SXT, 33.5% (2.2 to 71.0%). Resistance in non-OECD countries was higher, with resistance to TET 80.0% (59.7 to 95.3%); to AM, 67.2% (45.8 to 84.9%); and to SXT, 61.4% (no CI given) . Our study confirmed that resistance proportions are high in Thailand, a non-OECD country, with resistance proportions of 80% to TET, 72% to AM, and 66% to SXT in one-year old infants. One reason for the high prevalence of antibiotic resistance in Thailand healthy children may lie in wide use of antibiotics, which are available at grocery stores and retail shops without prescription . Moreover, high proportions of antibiotic resistance among bacteria have been found in various environmental sources in Thailand, including farms, water, and food . Additionally, extensive use of antibiotics in livestock worsens the situation by transmission of resistant strains to humans via the food chain [34, 35] . The prevalence of carriage of third-generation cephalosporine-resistant E. coli in the present study (both among infants and parents) was rather low compared with other regional investigations [18, . Fig. 4 Minimum-spanning tree of the 19 transmitted persistent (yellow), persistent transmitted (red), and control strains based on allelic profiles of the respective MLST ST. Each dot represents an allelic profile and the numbers on connecting lines display the number of differing alleles in a pairwise comparison. The dots are named with the isolate IDs and colored according to their assumed epidemiology / origin Luvsansharav et al. showed local differences in prevalence of ESBL-producing E. coli in healthy individuals in different provinces in Thailand varying between 32 and 53.9% . In the present study resistance against the third-generation cephalosporine ceftriaxone was found in only 20%. We did not perform a selective screening for third-generation cephalosporine-resistant E. coli using ESBLchromogenic media or MacConkey agar supplemented with a third-generation cephalosporine. This might have resulted in a low sensitivity. In the present study the most frequently detected resistance phenotypes were single resistance against tetracycline Table 3 Univariate analysis of determinants associated with prevalence of resistant E. coli in meconium (TET) (9.7%) and quadruple resistance against AM, TET, TMP, and SXT (21.5%). The high proportion of TET resistance is of interest when considering that TET is contraindicated in children. As persistence of resistant E. coli in children and transmission from parents to children was observed in only 6% each, we infer that in our study location, northern Thailand, a reservoir of resistance genes exists, with transmission mainly via horizontal gene transfer. This inference is supported by the finding that different MLST types were found in persistent, transmitted, and control strains. These resistant strains were detected soon after birth and resistance proportions also increased during the first year of life suggesting that parents and the environment are likely to be the main reservoir for these resistance genes. Another hypothesis, which was not investigated in the present study, is that resistant strains have likely been transmitted from the environment or from health-care-workers to the neonate or to the infant. However, exposure time was short when considering that meconium is usually passed within the first 48 h after birth. Both antibiotic use and day-care center exposure have been described as risk factors for the acquisition of resistant E. coli strains in neonates and young children [40, 41] . We found no statistically significant association between antibiotic use and occurrence of resistant E. coli. However, that most children who received antibiotic therapy did so aged between 6mo and 1 yr., and that at 6mo the prevalence of antibiotic resistance was already above 80%, makes it difficult to draw conclusions. In the present study no data on the attendance of day-care centers were collected. However, attendance of day care centers is common in Thailand and thus it can be assumed that this also had an influence on transmission and colonization with resistant E.coli in the present study. Several factors have been proposed as influencing the composition of the intestinal microbiota of infants. In the KOALA Birth Cohort Study of 1032 infants aged 1mo in the Netherlands, mode of delivery, type of infant feeding (breast feeding versus formula feeding), gestational age, and antibiotic use were the most important determinants for development of intestinal flora . In the present study we also found significant lower prevalence of resistant E. coli in meconium in children born by caesarian section compared to vaginal delivery. This is consistent with the findings of previous studies showing that delivery mode affects the direct transmission of initial bacteria from mother to newborns [43, 44] . In contrast, birth in the rural hospital was associated with an increased risk of early colonization with resistant E. coli strains, antibiotic usage by the mother during pregnancy showed to be protective. We can only speculate about the reasons for this. Less hygienic standards in rural hospitals might be an explanation for this. Some studies showed placental transfer of antibiotics such as erythromycin in humans . This might be a reason for delayed colonization with intestinal resistant bacteria in neonates. However, the reason why maternal antibiotic usage was protective in early colonization with resistant E. coli in the present study is not clear and needs further evaluation. We did not find that feeding behavior or antibiotic usage of the child affected gut colonization with resistant E. coli. Other determinants associated with prevalence of resistant E. coli in meconium like siblings or pets, were also not identified as significant in our study. We are aware that our study has several limitations. First, parents were taught by nurses how to collect and to store stool samples and how to keep the samples cooled during transport to the laboratory. Nevertheless, mistakes in sample handling by parents cannot be excluded. Furthermore, sample collection was initially planned at ages 4mo and 6mo. A flood in northern Thailand in 2011 isolated some study participants for several weeks. The 4mo and 6mo sample data therefore were pooled. Third, bacterial strains can be excreted intermittently. Thus by taking only one stool sample per time point we cannot exclude to have missed resistant E. coli. Furthermore, we cannot exclude that detection of a certain strain was influenced by our sampling processing protocol (including the colony picking) or by any unknown factors. Thus, our conclusions on transmission and persistence have to be interpreted with caution. Moreover, antibiotic use in infants during the first year of life may have influenced the diagnostic sensitivity of the culture method. However, resource limitations precluded testing of all E. coli cultured from stool samples. Another main limitation is the limited number of study participants which decreases the power of our study and thus the ability to detect further determinants associated with development of resistance.
Conclusion Antibiotic-resistant E. coli was found at high proportions in meconium of healthy Thai neonates (33%) in the present study. This value increased during the first year of life until it almost matched the value among adults. Vertical transmission from parents to their children was detected in only 6% of subjects. We speculate that a high resistance proportion in Thai infants may reflect horizontal gene transfer from reservoirs of resistance genes in parents, healthcare workers, and the environment. We further hypothesize that in order to prevent further increases of resistance, better infection control measures and cautious use of antibiotics are mandatory. Fig. 1 1 Fig. 1 Flowchart of stool sample collection over the one-year study period. Initially 169 families were included. 27 were excluded due to exclusion criteria (> 1 missing stool sample) or loss of follow-up
Fig. 2 2 Fig.2Prevalence of carriage of resistant E. coli resistant to tetracycline (TET), ampicillin (AM), co-trimoxazole (SXT), and cefazoline (CEF) in fathers, mothers, meconium of newborns, and infants at 2wk, 2mo, 4 -6mo, and 1y
Table 1 1 Demographic characteristics of study participants (n = 142) Number Proportion (%) Gender Males 75 52.8 Females 67 47.2 Week of gestation Preterms (<37wk) 20 14.1 Birth mode Vaginal 110 77.5 Caesarean-section 32 22.5 Birth complication of newborn Yes 8 5.6 Hospital Tertiary urban hospital 90 63.4 Urban birth clinic 31 21.8 Rural hospital 21 14.8 Antibiotic usage during pregnancy Yes 30 21.1 Siblings Yes 57 42.5 Pets Yes 67 47.2 Exclusively breast feeding at 48 h 95 66.9 2wk 109 76.8 2 m 106 74.6 4-6 m 77 54.2 1y 0 0 Antibiotic usage during 1y < 48 h 9 6.3 < 2wk 13 9.2 < 2 m 21 14.7 < 4-6 m 29 20.4 1y 54 38.0 First appearance of AB resistant E. coli Meconium 49 34.5 < 2wk 89 62.7 < 2 m 113 79.6 < 4-6 m 132 93.0 1y 142 100.0
Table 2 2 Numbers (n) of samples containing resistant E. coli and numbers of detected resistant E. coli strains in parents and infants during the first year of life TET CEF (%) (%) 87% 37% 87% 29% 30% 8% 39% 13% 46% 12% 65% 22% 80% 35% CRO (%) 17% 12% 3% 5% 5% 7% 13% CIP TZP (%) (%) 33% 1% 24% 0% 6% 0% 10% 0% 8% 0% 18% 0% 27% 3% FOX (%) 6% 2% 3% 1% 1% 4% 2% MEC (%) 7% 3% 2% 4% 8% 7% 9% CPD (%) 26% 16% 3% 7% 6% 10% 18% CXM (%) 33% 18% 4% 7% 7% 12% 17% GM FOT ETP AMC (%) (%) (%) (%) 25% 1% 0 (0) 6% 17% 0 (0) 0 (0) 3% 7% 0 (0) 0 (0) 1% 5% 0 (0) 0 (0) 1% 7% 1% 0 (0) 4% 16% 3% 0 (0) 7% 27% 1% 0 (0) 8% TMP (%) 71% 62% 19% 30% 39% 50% 65% CN SXT AM FM (%) (%) (%) (%) 24% 70% 84% 6% 13% 62% 87% 5% 4% 20% 25% 1% 7% 24% 33% 1% 8% 39% 49% 1% 13% 45% 72% 3% 18% 66% 72% 1% n detected resistant E. coli strains 295 263 65 71 99 198 232 n samples containing resistant E. coli (%) 131 (94) 135 (95) 47 (33) 70 (51) 84 (59) 111 (83) 131 (92) n samples 140 142 142 138 142 134 142 Population Follow-Up Event Fathers 48 h Mothers 48 h Infants 48 h Infants 2wk Infants 2mo Infants 4-6mo Infants 1y
|
10.1080/14786441308634993
|
public-domain
| null | null |
openalex
|
supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Downloaded by [Duke University Libraries] at 11:53 30 July 2012 effect. However, later measurements which they made after the tube had been differently treated led to a higher value of b, and the investigation referred to is not yet completed, so that the difference in the value of ~b given by the two methods may not be significant. In concluding we wish to correct an erroneous statement on p. 643 of the former paper. Maturer consideration has led us to conclude that the small systematic error referred to in the last paragraph but two makes the measured value of ~b too large at high temperatures and not too small as is there stated. In consequence we do not now regard the suggested increase of ~ with 8 as definitely established by the experiments. They are, in fact, insufficiently accurate for the purpose. We are glad to be able to take this opportunity of thanking Messrs. W. P. Schenck and W. R. Wensley, who took most of the observations under our direction. Palmer Physical Laboratory, Princeton~ N.J. w 1. General Assumptions. F OLLOWING the theory of Ruther[ord, we shall assume that the atoms el the elements consist of a positively charged nucleus surrounded by a cluster of electrons. The nucleus is the seat of the essential part of the mass of the atom, and has linear dimensions exceedingly small compared with the distances apart of the electrons in the surrounding cluster. As in the previous paper, we shall assume that the cluster of electrons is formed by the successive binding by the nucleus of electrons initially nearly at rest, energy at the same time being radiated away. This will go on until, when the total negative charge on the bound electrons is numerically equal to the positive charge on the nucleus, the system will be neutral and no longer able to exert sensible forces on electrons at distances from the nucleus great in comparison with the dimensions of the orbits of the bound electrons. We may regard the formation of helium from r rays as an observed example of a process of this kind, an a particle on this view being id6ntical with the nucleus of a helimn atom. On account of' the small dimensioi~s of the nucleus, its internal structure will not be of sensible influence on the constitution of the cluster of electrons, and consequently will have no effect on the ordinary physical and chemical properties of the atom. The latter properties on this theory will depend entirely on the total charge and mass of the nucleus; the internal structure of' the nncleus will be of influence only on the phenomena of radioactivity. From the result of experiments on large-angle scattering o~ a-rays, Rutherford* found an electric charge on the nucleus corresponding per atom to a number of electrons approximately equal to half the atomic weight. This result seems to be in agreement with the number of electrons per atom calculated from experiments on scattering of RSntgen radiation t. The total experimental evidence supports the hypothesis :~ that the actual nmnber of electrons in a neutral atom with a few exceptions is equal to the nmnber which indicates the position of the corre~,~ponding element in the series oi ~ elements arranged in order of increasing atomic weight. For example on this view, the atom of oxygen which is the eighth element of the series has eight electrons and a nucleus carrying eight unit charges. We shall assume that the electrons are arranged at equal angular intervals in coaxial rings rotating round the nucleus. In order to determine the frequency and dimensions of the rings we shall use the main hypothesis of the first paper, viz. : that in the permanent state of an atom the angular momentum of every electron round the centre el: its orbit is to the universal value ~!~, where h is Planck's equal constant. We shall take as a condition of stability, tha~ the ~otal energy of the system in the configuration in question is less than in any neighbouring configuration satisfying the same condition of the angular momentum of the electrons. If the charge on the nucleus and the number of electrons in the different rings is known, the condition in regard to the angular momentum of the electrons will, as shown in w 2, cmnpletely determine the configuration of the system, i. e., the frequency of revolution and the linear dimensions of the rings. Corresponding to different distributions of the Dr. ~q. Bohr on the Constitution electrons in the rings, however, there will, in general, be more than one configuration which will satisfy the condition of the angular momentmn together with the condition of stability. In w 3 and w 4 it will be shown that, on the general view of the formation of the atoms, we are led to indications of the arrangement of the electrons in the rings which are consistent with those suggested by the chemical properties of the corresponding element. In w 5 it will be shown that it is possible from the theory to calculate the minimum velocity of cathode rays necessary to produce the characteristic Riintgen radiation from the element, and that this is in approximate agreement with the experimental values. In w 6 the phenomena of radioactivity will be briefly considered in relation to the theory. If F is known, the dimensions and frequency of the corresponding orbit are simply determined by ( 1 ) and ( 2 ). For a Downloaded by [Duke University Libraries] at 11:53 30 July 2012 ring of n electrons rotating round a nucleus of charge ~qe we have (comp. Part [., p. 20) s=u--I $7i" F = N--so where .~-----4 Z cosec -n-" e=l 'The wdues for s~, from n=l to n=16 are given in the table on p. 482. For systems consisting of nuclei and electrons in which the first are at rest and the latter move in circular orbits with a velocity small compared with the velocity of light, we have shown (see Part I., p. 24) that the total kinetic energy of the electrons is equal to the total amount of energy emitted (luring the formation of the system from an original configuration in which all the particles are at rest and at infinite distances from each other. Denoting this amount of energy by W, we consequently get Putting in (1), (2), and (3) e=4"7.10 -1~ e =5"31 1017, and h=6"5 . 10 -27 we get a=0"55. 10-8F -1, v=2"1 . 10SF, w=6"2 . 1015F 2"} and ~ (4) W=2"0 . 10-nZF ~. In neglecting the magnetic forces due to the motion of the electrons we have in Part I. assumed that the velocities of the particles are small compared with the velocity of light. The above calculations show that for this to hold, F must be small compared with 150. As will be seen, the latter condition will be satisfied for all the electrons in the atoms of elements of low atomic weight and for a greater part of the electrons contained in the atoms of the other elements. If the velocity of the electrons is not small compared with the velocity of light, the constancy of the angular momentum no longer involves a constant ratio between the energy and ttle frequency of revolution. Without introducing new assumptions, we cannot therefore in this case determine the configuration of tile systems on the basis of the considerations in Part I. Considerations given later suggest, however, that the constancy of the angular momentum is the principal condition. Applying this condition ibr velocities 2K2 Downloaded by [Duke University Libraries] at 11:53 30 July 2012 Dr. N. Bohr on the Co~stit~tion not small compared with the velocity of light, we get the same expression for v as that given by (1), while the quantity m in the expressions for a and o~ is replaced by ~/(l__v~/~2), and in the expression for W by As stated in Part I., a calculation based on the ordinary mechanics gives the result, that a ring of electrons rotating round a positive nucleus in general is unstable for displacements of the electrons in the plane of the ring. In order to escape from this difficulty, we have assumed that the ordinary principles o~ mechanics cannot be used in the discussion of the problem in qnestion, any more than in the discussion of the connected problem of the me~-hanism of binding of electrons. We have also assumed that the stability for such displacements is secured through the introduct]'on of the hypothesis of the universal constancy of the angular momentum of the electrons. As is easily shown, the latter assmnption is included in the condition of stability in w 1. Consider a ring of electrons. rotating round a nucleus, and assume that the system is i~l dynamical equilibrimn and that the radius of the ring is a0, the velocity of the electrons re, the total kinetic energy T 0, and the potential energy P0-As shown in Part I. (p. 21) we have Po=--2To. Next consider a conliguration of the system in which the electrons, under influence of extraneous forces, rotate with the same angular momentum round the nucleus in a ring of radius a=aao. In this case we have p___ 1po, and on account of the uniformity of the angular We see that the total energy of the new configuration is greater than in the original. According to the condition of stability in w 1 the system is consequently stable for the displacement considered. In this connexion, it may be remarked that in Part I. we have assumed that the frequency of radiation emitted or absorbed by the systems cannot be determined from the frequencies of vibration of the electrons in the plane of the orbits, calculated by help of the ordinary Downloaded by [Duke University Libraries] at 11:53 30 July 2012 mechanics. We have, on the contrary, assumed that the frequency of the radiation is determined by the condition J~v= E, where v is the frequency, h Planck's constant, and E the difference in energy corresponding to two different " stationary" states of the system. In considering the stability of a ring of electrons rotating round a nucleus for displacements of the electrons perpendicular to the plane of the ring, imagine a configuration of the system in which the electrons are displaced by (~zl, 8z~ .... 8z,, respectively, and suppose that the electrons, under influence of extraneous s rotate in circular orbits parallel to the original plane with the same radii and the same angular momentum round the axis of the system as before. The kinetic energy is unaltered by the displacement, and neglecting powers of the quantities 8zl, 9 9 9 9 8z,~ higher than the second, the increase of the potential energy of the system is given by ] where a is the radius of the ring, Ne the charge on the nucleus, and n the nmnber of electrons. According to the condition of stability in w 1 the system is stable for the displacements considered, if the above expression is positive for arbitrary values of 8zl, .... 8z,,. By a simple calculation it can be shown that the latter condition is equivalent to the condition N >?,,,o -p,,, .,, . ..... (5) where m denotes the whole number (smaller than n) for which :~ cos 2k cosec3 s~r has its smallest value. This condition is identical with the condition of stability for displacements of the electrons perpendicular to the plane of the ring, deduced by help of ordinary mechanical considerations *. A suggestive illustration is obtained by imagining that the displacements considered are produced by the effect of extraneous forces acting on the electrons in a direction parallel to the axis of the ring. If the displacements are produced infinitely slowly the motion of the electrons will at any moment be parallel to the original plane of the ring, and the angular momentum of each of the electrons round 9 Comp. ft. W. Nicholson, Month. Not. Roy. Astr. Soc. 72. p. 52(1912) . Downloaded by [Duke University Libraries] at 11:53 30 July 2012 the centre of its orbit will obviously be equal to its original value; the increase in the potential energy of the system will be equal to the work done by the extraneous forces during the displacements. From such considerations we are led to assume that the ordinary mechanics can be used i~1 calculating the vibrations of the electrons perpendicular to the plane of the ring--contrary to the ease of vibrations in the plane of the ring. This assumption is supported by the apparent agreement with observations obtained by ~qicholson in his theory of the origin of lines in the spectra of the solar corona and stellar nebulae (see Part I. pp. 6 & 23) . In addition it will be shown later that the assumption seems to be in agreement with experiments on dispersion. The following We see from the table that the number of electrons which can rotate in a single ring round a nucleus of charge e increases only very slowly for increasing ~ ; for N=20 the maximum value is n=10; for ~T=40, n=13; for N----60, n=15. We see, further, that a ring of n electrons cannot rotate in a single ring-round a nucleus of charge ne unless n<8. In the above we have supposed that the electrons move under the influence of a stationary radial force and that their orbits are exactly circular. The first condition will not be satisfied if we consider a system containing several rings of electrons which rotate with different frequencies. If, however, the distance between the rings is not small in comparison with their radii, and if the ratio between their frequencies is not near to unity, the deviation from circular orbits may be very small and the motion of the electrons to a close approximation may be identical with that obtained on the assumption that the charge on the electrons is uniformly distributed along the circumference of tile rings. If the ratio between the radii of the rings is no~ Downloaded by [Duke University Libraries] at 11:53 30 July 2012 near to unity, the conditions of stability obtained on this aasumption may also be considered as sufficient. We have assumed in w 1 that the electrons in the atoms rotate in coaxial rings. The calculation indicates that only in the case of systems containing a great number of electrons will the planes of the rings separate ; in the case of systems containing a moderate nmnber of electrons, all the rings will be situated in a single plane through the nucleus. For the sake of brevity, we shall therefore here only consider the latter case. Let us consider an electric charge E uniformly distributed along the circumference of a circle of radius a. At a point distant z from the plane of the ring~ and at a distance r from the axis of the ring, the electrostatic potential is given by where Q(~)= ~sin 4 a(K(2a)-cot ~K'(2a)). 7t The corresponding force perpendicular to the plane of the ring at a distance ~' from the centre of the ring and at a small distance Sz ~rom its plane is given by ~U Ee3"R a, on an electron in the rth ring ~r:F,., where the summation is to be taken over all the rings except the one considered. If we know the distribution of the electrons in the different rings, from the relation (1) on p. 478, we can, by help of t~he above, determine as, as,.... The calculation can be made by successive approximations, starting from a set of values for the at's, and from them calculating the F's, and then redetermining the a's by the relation (1) which gives F~F* = a-~a~ =tan s (a,. ~),, and so on. As in the case of a single ring it is supposed that the systems are stable for displacements of the electrons in the plane of their orbits. In a calculation such as that on p. 480, the interaction of the rings ought strictly to be taken into account. This interaction will involve that the quantities F are not constant, as for a single ring rotating round a nucleus, but will vary with the radii of the rings ; the variation in F, however, if the ratio between the radii or' the rings is not very near to unity, will be too small to be of influence on the result of the calculation. Considering the stability of the systems for a displacement of the electrons perpendicular to the plane of the rings, it is necessary to distinguish between displacements in which the centres of gravity of the electrons in the single rings are unaltered, and displacements in which all the electrons inside the same ring are displaced in the same direction. The condition of stability for the first kind of displacements is given by the condition ()5 on .13 481, if for every, ring .we replace ~N by a quantity G~ determined by the condition If all the electrons in one of the rings are displaced in the same direction by help of extraneous forces, the displacement will produce corresponding displacements of the electrons in the other rings ; and this interaction will be of influence on the stability. For example, consider a system of m concentric rings rotating in a plane round a nucleus of charge Ne, and let us assume that the electrons in the different rings are displaced perpendicular to the plane by ~zl, Sz~,.... 6z~ respectively. With the above notation the increase in the potential energy of the system is given by The condition of stability is that this expression is positive for arbitrary values of 3q,....Sz,~. This condition can be worked out simply in the usual way. It is not of sensible influence compared with the cmldition of stability tbr the displacements considered above, except in cases where the system contains several rings of few electrons. The following Table , containing the values of Q(~) and R(a) for every fifth degree from a=20 ~ to a=70 ~ gives an estimate of the order of magnitude of these functions :-- to neutralize approximately the effect of a part of the charge on the nucleus corresponding to the number of electrons on the ring. The values of R(a) show that, the effec~ of outer rings on the stability of inner--though greater than the ei~ect on the dimensions--is small, but that unless the ratio between the radii is very great, the effect of inner rings o~ the stability of outer is considerably greater than to neutralize a corresponding part of the charge of the nucleus. The maximum number of electrons which the innermost ring can contain without being unstable is approximately equal to that calculated on p. 482 for a single ring rotating round a nucleus. For the outer rings, however, we get considerably smaller numbers than those determined by the condition (5) if we replace Ne by the total charge on the nucleus and on the electrons of inner rings. If a system oi" rings rotating round a nucleus in a single plane is stable for small displacements of the electrons perpendicular to this plane, there will in general be no stable configurations of the rings, satisfying the condition of the constancy of the angular momentum of the electrons, in which all the rings are not situated in the plane. An exception occurs in the special case of two rings containing equal numbers of electrons ; in this case there nmy be a stable configuration in which the two rinRs have equal radii and rotate in parallel planes at equal distances from the nucleus~ the electrons in the one ring being situated iust opposite the intervals between the electrons in the other ring. The latter configuration, however, is unstable if the configuration in which all the electrons in the two rings are arranged in a single ring is stable. w 3. Constitution of Atoms containing very.few Electrons. As stated in w 1, the condition of the universal constancy of the angular momentmn of the electrons, together with the condition of stability, is in most cases not sufficient to determine completely the constitution of the system. On the general view of formation of atoms, however~ and by making use of the knowledge of the properties of the corresponding elements, it will be attempted, in this section and the next, to obtain indications of what configurations of the electrons may be expected to occur in the atoms. In these considerations we shall assume that the number ot[ electrons in the atom is equal to the number which indicates the position of the corresponding element in the series of elements arranged in order of increasing atomic weight.
Downloaded by [Duke University Libraries] at 11:53 30 July 2012 Exceptions to this rule will be supposed to occur only at such places in the series where deviation from the periodic law of the chemical properties of the elements are observed. In order to show clearly the principles used we shall first consider with some detail those atoms containing very few electrons. For sake of brevity we shall, by the symbol N(nl, n2 .... ), refer to a plane system of rings of electrons rotating round! a nucleus of charge Ne, satisfying the condition of the angular momentum of the electrons with the approximation used in w 2. nl, n~ .... are the numbers of electrons in the rings, starting from inside. By al, a~,.,, and eel, eoe,... we shall denote the radii and frequency of the rings taken in the same order. The total amount of energy W emitted: by the formation of the system shall simply be denoted by )]. N = 1. I]~/drogen. In Part I. we have considered the binding of an electron by a positive nucleus of charge e, and have shown that it is possible to account for the Balmer spectrum of hydrogen on the assumption of the existence of a series of stationary states in which the angular momentum of the electron round h the nucleus is equal to entire multiples of the value ~, where h is Planck's constant. Tile formula found for the frequencies. of the spectrum was For the permanent state of a neutral hydrogen atom we get from the formula (1) and (2) in w 2, putting F= 1, 4~r2e4m ~ . h2 =0"55 10 -s, ~o=--h~--=(i-Z 1015 , 1 (1). a = 4~r~e:~ 9 ~Ofr~e41?l W= --=2"0.10 -11 . h ~ These values are of the order of magnitude to be expected. For W --we get 0'043, which corresponds to 13 volts; the e value ibr the ionizing potential of a hydrogen atom, calculated by Sir J. J. Thomson from experiments on positive rays, is iX volts ~. No other definite data, however, are available for hydrogen atoms. For sake of brevity, we shall ia the following denote the values for a, ~o, and W corresponding to the configuration 1(1) by a0, ~o0 and ~g0. At distances from the nucleus, great in comparison with a0, the system ](1) will not exert sensible forces on free electrons. Since, however, the configuration : 1(2) a=1"33 a0, 0)=0"563 co0, W=I'13Wo, corresponds to a greater value for W than the configuration 1(1), we may expect that a hydrogen atom under certain conditions can acquire a negative charge. This is in agreement with experiments on positive rays. Since W[l(3)] is only 0"54, a hydrogen atom cannot be expected to be able to acquire a double negative charge. N = 2. Helh~m. As shown in Part I., using the same assumptions as for hydrogen, we must expect that during the binding of an electron by a nucleus of charge 2e, a spectrmn is emitted, expressed by 27r2me ' 1 1)) This spectrmn includes the spectrum observed by Pickering in the star ~* Puppis and the spectra recently observed by Fowler in experiments with vacuum tubes filled with a mixture of hydrogen and helium. These spectra arc generally ascribed to hydrogen. For the permanent state of a positively charged helium atom, we get 2 (1) a = 89 ~ = 4~0, W= 4W0. At distances from the nucleus great compared with the radius of the bound electron, the system 20) will, to a close approximation, act on an electron as a simple nucleus of charge e. For a system consisting of two electrons and a nucleus of charge 2e, we may therefore assume the existence of a series of stationary states in which the clectron most lightly bound moves approximately in the same way as the electron in the stationary states of a hydrogen atom. Such an assumption has aheady been used in Part I. in an attempt to explain the appearance of R)'dberg's constant in the formula for the line-spectrum of any element. We can, however, hardly assume the existence of a stable configuration in which the two electrons have the "same angular momentum round the nucleus and move in different orbits, the one outside the o~her. In such a configuration ttle electrons would be so near to each other that the deviations t~rom circular orbits would be very great 9 For the permanent state of a neutral helium atom, we shall therefore adop~ the configuration 2(2) a-~0"571 ao, (o =3"06 co 0, W= 6"13 W0. Since W[2(2)~ -w[~(~)~ = 2.13 w0, we see that both electrons in a neutral helium atom are'more firmly bound than the electron in a hydrogen atom. Using the values on p. 4~8, we get 2"13 ~=27 volts and 2"13~=6"6 . 101~ ~1_. 9 e sec. ~ these values are of the same order of magnitude as the value observed for the ionization potential in helium, 20"5 volt*, and the value for the fi'equency of the ultra-violet ab-.~orption in helium determined by experiments on dispersion 15 1 5"9.10 ~ r The frequency in question may be regarded as corresponding to vibrations in the plane of the ring (see p. 480). The frequency of vibration of the whole ring perpelldicular to the plane, calculated in the ordinary way (see p. 482), is * J. Franck u. G. Hertz, Verb. d. JDeutsch. -Phys. Ges. xv. p. 34 (1913). t C. and M. Cuthbertson, Proc. Roy. Soc. A. lxxxiv, p. 13 (1910) . (In a previous paper (Phil. l~lag. Jan. 1913) the author took the values for the refractive index in helium, given by 5I. and C. Cuthbertson, as corresponding to atmospheric pressure; these values, however, refer to double atmospheric pressure. Consequently the value there given for the number of electrons in a helium atom calculated fi'om Drude's theory has to be divided by 2.) Downloaded by [Duke University Libraries] at 11:53 30 July 2012 Dr. N. Bohr on the Constltutlor~ given by z,=3"'27 co 0. The fact that the latter frequency is great compared with t hat~ observed might explain tha~ the nmnber of electrons in a helium atom, calculated by help of Drude's theory from the experiments on dispersion, is only about two-thirds of the nmnber to be expected. (Using e 1017 --=5"31 . the value calculated is I'2.) ]~'or a configuration of a helium nucleus and three electrons} we get 2(3) a----0"703a o, co=2"02 ~o0, W=6"07 W0. Since W for this configuration is smaller allan for the coufiguration 2(2), the theory indicates that a helium atom cannot acquire a negative charge. This is in agreement with experimental evidence, which shows that helimn atoms have no "affinity" for free electrons *. In a later paper it will be shown that the theory offers a simple explanation of the marked difference in the tendency of hydrogen and helimn atoms to combine into molecules.
N = 3. Lithium. In analogy with the eases of hydrogen and helimn we must expect that during the binding of an electron by a nucleus of charge 3e, a spectrum is emitted, given by On account of the great energy to be spent in removing all the electrons bound in a lithium atom (see below) the spectrmn considered cau only be expected to be observed in extraordinary cases. In a recent note Nicholsont has drawn attention to the fact that in the spectra of certain stars, which show the Pickering spectrum with special brightness, some lines occur the frequencies of which to a close approximation can be expressed by the forumla v----K(~ -~), ( -~) where K is the same constant as in the Balmer spectrum of hydrogen. From analogy with the Balmer-and Pickeringspectra, Nicholson has suggested ghat the lines in question are due to hydrogen. It is seen that the lines discussed by Nicholson are given by the above formula if we put T2=6. The lines in question correspond to vl = 10,13, and 14 ; if we for T,~ = 6 put ~1 = 9, 12 and 15, we get lines coinciding with lines of the ordinary Balmer-spectrum of hydrogen. If we in the above formula put -r=l, 2, and 3, we get series of lines in the ultra-violet. If we put ~'2=4 we get only a single line in visible spectrum, -viz.: for rl=5 which gives t,=6"662.101~, or a wave-length X=4503.10 -s era. closely coinciding with the wave-length 450~. 10 -s cm. of one or' the lines of unknown origin in the table quoted by Nieholson. In this table, however, no lines occur corresponding to ~'2= 5. For the permanent state of a lithium atom with two positive charges we get a configuration 3(1) a= 89 to = 9a~o, w=gw o . The probability of a permanent configuration in which ~wo electrons move in different orbits around each other must for lithium be considered still less probable than for helium, as the ratio between the radii of the orbits would be still nearer to unity. For a lithium atom with a single positive charge we shall, therefore, adopt the configuration: 3(2) a=O'36tao, to= 7"56 t%, W=15"13Wo. Since W[3(2)J-W[3(1)J=6"13W0, we see that the first two electrons in a lithium atom are very strongly bound compared with the electron in a hydrogen atom; they are still more rigidly bound than the electrons in a helium atom. From a consideration of the chemical properties we should expect the following configuration for the electrons in a neutral lithium atom : will not arrange themselves in a single ring and form the system : 3 (3) a=0"413 a0, to=5"87 too, W= 17"6l W0, although W for this configuration is greater than for 3(2, 1). Since W[3(2,1)]--W[3(2)] =0"89 W0, we see that the outer electron in the configuration 3 (2, i) is bound even more lightly than the electron in a hydrogen atom. The difference in the firmness of the binding corresponds to a difference of 1"4 volts in the ionization potential. Amarked difference between the electron in hydrogen and the outermost electron in lithium lies also in the greater tendency of the latter electron to leave the plane of the orbits. The quantity G considered in w 2, which gives a kind of measure for the stability ibr displacements perpendicular to this plane, is thus for the outer electron in lithium only 0"55, while for hydrogen it is 1. This nmy have a bearing on the explanation of the apparent tendency of lithimn atoms to take a positive charge in chemical combinations with other elements. For a possible negatively charged lithimn atom we may expect the configuration : a=0'362 ao to= 7"64 too W----16'16 We.
3(2,2) a=l"516ao to=0"436% It should be remarked that we have no detailed knowledge of the properties in the atomic state, either for lithium or hydrogen, or for most of the elements considered below.
N =4. BerSlium. For reasons analogous to those considered for helium and lithium we nmy for the formation of a neutral beryllium atom assume the following stages : correspond to less values for the total energy than the configurations 4 (2, 1) and 4 (2.2). From analogy we get further for the configuration of a possible negatively charged atom, al=O'263ao ~ol= 14"51 We 4(2 3~ a2=0'803a0 eel= 1"55 ~o 0 W=33"66 W0. Comparing the outer ring of the atom considered with the ring of a helimn atom, we see that the presence of the inner ring of two electrons in the beryllimn atom marked Iy changes the properties of the outer ring; partly because the outer electrons in the configuration adopted for a neutral beryllium arena are more lightly bound than the electrons in a hetimn atom, and partly because the quantity G, which for heliu,n is equal to 2, for the outer ring in the configm-ation 4 (2,2) is only equal to 1"12. Since W~4 (2,3)J--W[4(2,2)J =0'05 W0, the beryllimn atom will further have a definite, although very small affinity for free electrons. w 4. Atoms co~taiui~g greater numbers of electrons. Froln the examples discussed in the former section it will appear that the problem of the arrangement of the electrons in the atoms is intimately connected with the question of the confluence of two rings of electrons rotating round a nucleus outside each other, and satisfying the condition of the universal coustancy of the angular momentum. Apart from the necessary conditions of stability for displacements of the electrons perpendicular to the plane of the orbits, the present theory gives very little information on this problem. It se~ms, however, possible by the help of simple considerations to throw Some light on the question. Let us consider two rings rotating round a nucleus in a single plane, the one outside the other. Let us assume that the electrons in the one ring act upon the electrons in the other as if the electric charge were uniforlnly distributed along flhe circumference of the ring, and that the rings with Ibis approximation satisfy the condition of the angular momentum of the electrons and of stability for displacements perpendicular to their plane. Now suppose that, by help of suitable imaginary extraneons forces acting parallel to the axis of the rings, we pull the inner ring slowly to one side. During this process, on account of the repulsion from the inner ring, the outer will move to the opposite side of the original plane of the rings. Phil. MaR. S. 6. Vol. 26. No. 15:3. Seyt. 1913 .
Dr. ~N. Bohr on the Constitution During the displacements of the rings the angular momentun~ of the electrons round the axis of the system will remain constant, and the diameter of the inner ring will increase while that of the outer will diminish. At the beginning of the displacement the magnitude o~ the extraneous forces to be applied to the original inner ring will increase but thereafter decrease, and at a certain distance between tile plane ,of the rings the system will be in a configuration of equilibrium. This equilibrium, however, will not b~ stable, if we let the rings slowly return they will either reach their original position, or they will arrive at a position in which the ring, which originally was the outer, is now the inner, and vice versa. If the charge of the electrons were uniformly distributed along the circumference of the rings, we could by 1he process considered at most obtain an interchange of the rings, hut obviously not a junction of them. Taking, however, the discrete distribution of the electrons into account, it can be shown that, in the special case when the number of electrons on the two rings arc equal, and when the rings rotate in the same direction, the rings will unite by the process, provided that the final configuration is stable. In this case the radii and the frequencies of the rings will be equal in the unstable configuration or" equilibrium mentioned above. In reaching this configuration the electrons in the one ring will further be situated just opposite the intervals between the electrons in the other, since such an arrangement will correspond to the smallest total energy. If now we let ~he rings return ~o their original plane, the electrons in the one ring will pass into the intervals between the electrons in the other, and form a single ring. Obviously the ring thus formed will satisfy the same condition of the angular momentum of the electrons as the original rings. if the two rings contain unequal nmnbers of electrons the system will during a process such as that considered behave very differently, and, contrary to the former case, we cannot expect that the rings will flow together, if by help of extraneous forces acting parallel to the axis of tim system they are displaced slowly from their original plane. It may in this connexion be noticed that the characteristic for the displacements considered is not the special assumption about the extraneous forces, but only the invariance cf the angular momentum of the electrons round the centre of the rings ; displacements of this kind take in the present theory ,~ similar position to arbitrary displacements in the ordinary mechanics.
Downloaded by [Duke University Libraries] at 11:53 30 July 2012 The above considerations may be taken as an indication that there is a greater tendency ibr the confluence of two rings when each contains the same number of electrons. Considering the successive binding of electrons by a positive nucleus, we conclude from this that, unless the charge on the nucleus is very great, rings of electrons will only join together if they contain equal numbers of electrons; and that accordingly the numbers of electrons on inner rings will only be 2, 4, 8, .... If the charge of the nucleus is very great the rings of electrons first bound, if few in number, will be very close together, and we must expect that the configuration will be very unstable, and that a gradual interchange of electrons between the rings will be greatly facilitated. This assumption in regard to the number of electrons in the rings is strongly supported by the fact that the chemic'll properties of the elements of low atomic weight vary with a period of 8. Further, it follows that the number of electrons on the outermost ring will always be odd or even, according as the total number of electrons in the atom is odd or even. This has a suggestive relation to the fact that the valency of an element of low atomic weight always is odd or even according as the number of the element in the periodic series is odd or even. For the atoms of the elements considered in the former section we have assumed that the two electrons first bound are arranged in a single ring, and, further, that the two next electrons are arranged in another ring. ]f N_ > 4 the configuration N(4)will correspond to a smaller v-alue for the total energy than the configuration N (2,2). The greater the value of ~ the closer will the ratio between the radii el the rings in the configuration ~ (2, 2) approach unity, and the greater will be the energy emitted by an eventual confluence of the rings. The particular member el the series of the elements for which the four innermost electrons will be arranged for the first time in a single ring cannot be determined from the theor). From a consideration of the chemical properties we can hardly expect that it will have taken place befbre boron (N----5)or carbon (N~-6), on account of the observed trivalency and tetravalency respectively of these elements; on the other hand, the periodic system of the elements strongly suggests that already in neon (N----10) an inner ring of eight electrons will occur. Unless N~14 the configuration N (4, 4) corresponds to a smaller value for the total energy than the configuration N(8); already 2L2 Downloaded by [Duke University Libraries] at 11:53 30 July 2012 for N>__~10 the latter configuration, however, will be stable for displacements of the electrons perpendicular to the plane of their orbits. A ring of 16 electrons will not be stable unless N is very great; but in such a case the simple considerations mentioned above do not, apply. The confluence of two rings of equal nmnber of electrons, which rotate round a nucleus of charge Ne outside a ring of n electrons already bound, must be expected to take place more easily than t~he confluence of two similar rings rotating round a nucleus of charge (N--n)e ; for the stability of the rings for a displacement perpendicular to their plane wilt (see w 2) be smaller in the first than in the latter case. This tendency for stability to decrease for displacements perpendicular to the plane of the ring will be especially marked for the outer rings of electrons of a neutral atom. In the latter case we must expect the confluence of rings to be greatly facilitated, and in certain cases it ma 3 even happen that the nuinber of electrons in the outer ring may be greater than in the next, and that the outer ring may show deviations from the assumption of 1, 2, 4, 8 electrons in the rings, e. g. the configurations 5 (2,3) aud 6 (2,4) instead of tho configurations 5 (2, 2,1) and 6 (2,2,2). We shall here not discuss further the intricate question of the arrangement of' the electrons in the outer ring." In the scheme given below the number el electrons in this ring is arbitrarily put equal to the normal valency of' the corresponding element ; i. e. for electronegative and electropositive elements respectively the number of hydrogen atoins and twice the number of oxygen atoms with which one atom of the element combines. Such an arrangement of the outer electrons is suggested by considerations of atomic w)lmnes. As is well known, the atomic volume of the elements is a periodic function of the, atomic weights. If arranged in the usual way according to the periodic system, the elements inside the same cohmm have approxinmtely the same atomic volume, while this volmne changes considerably from one colmnn to another, being greatest for colunms corresponding to the smallest valency i and smallest for the greatest valency 4. An approximate estimate of the radius of the outer ring of a neutral atom can be obtained 1)y assmning that the total force due to the nucleus and the inner electrons is equal to that from a nucleus of ehargo he, where n is the number of electrons in the ring. Putting F=n-s,~ in the equation (1) on p. 478, and denoting the value of a for n=l by a0, we get for n=2, a=0"57a0; for n=3, a=0"41ao; and for n = 4, a = 0"33a0. Accordingly the arrangement cl~osen Downloaded by [Duke University Libraries] at 11:53 30 July 2012 for the electrons will involve a variation in the dimensions of the outer ring similar to the variation in the atomic volumes of the corresponding elements. It mus~, however, be borne in mind that the experimental determinations of atomic volumes in most cases are deduced from consideration of molecules rather than atoms. From the above we are led to the following possible scheme for the arrangement of the electrons'in light atoms:--1 ( 1 Without any fuller discussion it seems not unlikely that this constitution of the atoms will correspond to properties of the elements similar with those observed. In tile first place there will be a marked periodicity with a period of 8. Further, the binding of the outer electrons in every horizontal series of the above scheme will become weaker with increasing number of electrons per atom, corresponding to the observed increase of the eleetropositivo character for all increase of atomic weight of the elements in every single group of the periodic system. A cot'responding agreement holds for the variation of the atomic volumes. In the ease of atoms of higher atomic weight the simple assumptions used do not apply. A few indications, however, are suggested froln consideration of the variations in the chemical properties of the elements. At the end of ~ho 3rd period of 8 elements we lneet with the iron-group. This group fakes a particular position in the system of the elements, since it is the first time that elements of neighbonring atomic weights show similar chemical properties. This circmnstance indicates that the configurations of the electrons in the elements of this group differ only in the arrangement of the inner electrons. The fact that the period in the ebemicat properties of the elements after the iron-group is no longer 8, but 18, suggests that elements of higher atomic weight contain a recurrent configuration of 18 electrons in the innermost rings. The deviation from Downloaded by [Duke University Libraries] at 11:53 30 July 2012 2, 4, 8, 16 may be due to a gradual interchange of electrons between the rings, such as is indicated on p. 495. Since a ring of 18 electrons will not be stable the electrons may be arranged in two parallel rings (.see p. 486). Such a configuration of the inner electrons will act upon the outer electrons iu very nearly the same way as a nucleus of charge (--18)e. It might therefore be possible that with increase of N another configuration of the same type will be formed outside the first, such as is suggested by the presence of a second period of 18 elements. On the same lines, the presence of the group of the rare earths indicates that for still greater values of N another gradual alteration of the innermost rings will take place. Since, however, for elements of higher atomic weight than those of this group~ the laws connecting the variation of the chemical properties with the atomic weight are similar to those between the elements of low atomic weight~ we may conclude that the configuration of the innermost electrons will be again repeated. The theory, however, is not sufficiently complete to give a definite answer to such problems. w 5. Characteristic R6ntgen Radiation. According to the theory of emission of radiation given in Part I., the ordinary line-spectrum of an element is emitted during the reformation of an atom when one or more of the electrons in the outer rings are removed. In analogy it may be supposed that the characteristic RSntgen radiation is sent out during the settling down of the system if electrons in inner rings are removed by some agency, e.g. by impact of cathode particles. This view of the origin of the characteristic Riintgen radiation has been proposed by Sir J. J. Thomson*. Without any. special assumption in regard to the constitution of the radiation, we can from this view determine the minimum velocity of the cathode rays necessary to produce the characteristic RSntgen radiation of a special type by calculating the energy necessary to remove one of the electrons from the different rings. Eveu if we knew the numbers of electrons in the rings, a rigorous calculation of this minimum energy might still be complicated, and the result largely dependent on the assumptions used; for~ as mentioned in Part I., p. 19, the calculation cannot be performed entirely on the basis of the ordinary mechanics. We can) however) obtain very simply an approximate comparison * Comp. J. J. Thomson, Phil. ~[ag. xxiii, p. 456 (1912) . Downloaded by [Duke University Libraries] at 11:53 30 July 2012 with experiments if we consider the innermost ring and as a first approximation neglect the repulsion from the electrons in comparison with the attraction of the nucleus. Let us consider a simple system consisting of a bound electron rotating in a circular orbit round a positive nucleus oE charge No. From the expressions (1) on p. 478 we get for the velocity of the electron, putting F----N, v= 2he! N--2"l. 10sN. The total energy to be transferred to the system in order to remove the electron to an infinite distance from the nucleus is equal to the kinetic energy of the bound electron. If, therefore, the electron is removed to a great distance from the nucleus by impact of another rapidly moving electron, the smallest kinetic energy possessed by the latter when at a great distance fl'om the nucleus must necessarily be equal to the kinetic energy of the bound electron before the collision. The velocity of the free electron therefore must be at least equal to v. According to Whiddington's experiments * the velocity of cathode rays just able to produce the characteristic RSntgen radiation of the so-called K-type--the hardest type of radiation observed--from an element of atomic weight A is for elements from A1 to Se approximately equal to A. 10 ~ cm./sec. As seen this is equal to the above calculated value for v, if we put N = _A.
2 Since we have obtained approximate agreement with experiment by ascribing the characteristic R5ntgen radiation of the K-type to the innermost ring, it is to be expected that no harder type of characteristic radiation wlil exist. This is strongly indicated by observations of the penetrating power of 7 rays?. It is worthy of remark that the theory gives not only nearly the right value for the energy required to remove an electron from the outer ring, but also the energ.y required to remove an electron from the innermost ring. The approximate agreement between the calculated and experimental values is all the more striking when it is recalled that the energies required in the two cases for an element of atomic weight 70 differ by a ratio of 1000. In connexion with this it should be emphasized that the Putting in (4)F=N, we get for the diameter of the 1 innermost ring approximately 2a = N " 10-s cm. For N = 100 this gives 2a = 10-1~ a value which is very small in comparison with ordinary atomic dimensions but still very great compared with the dhnensions to be expected for the nucleus. According to l~utherford's calculation the dimensions of the latter are of the saule order of magnitude as 10-12 cm. w 6..Radioactive ]Dhenome~za. According to the present theory the cluster of electrons surrounding the nucleus is formed witii emission of energy, and the configuration is determined by the condition tbat the energy emitted is a maximum. The stability involved by these assmnptions seems to be in agreement with the general properties ot' matter. It is, however, in striking opposition to the t)henomena of radioactivity, and according to the theory the origin of the lat~er phenomena may therefore be sough~ elsewhere titan in the electronic distribution round the nucleus. A necessary consequence of Rutherford's theory of the structure of atoms is that the ~-particles have their origin in the nucleus. On the presen~ theory it seems also necessary that the nucleus is the sea~ of 'the expulsion of the high-speed /3-particles. In tile first place, the spontaneous expulsion of a/3-particle from the cluster of electrons surrounding the nucleus would be something quite tbreign to the assumed properties of the system. Further, the expulsion of an a-particle can hardly be expected to produce a lasting effect on the stability of the cluster of electrons. The effect of the expulsion will be of two different kinds. Partly the particle may collide with the bound electrons during its passing through the atom. This effect will be analogous to tha~ produced by bombardment of atoms of other substances by a-rays and cannot be expected to give rise to a subsequent expulsion of fl-rays. Partly the expulsion of the particle Downloaded by [Duke University Libraries] at 11:53 30 July 2012 will involve an alteration in the configuration of the bound electrons, since the charge remaining on the nucleus is different from the original. In order to consider the latter effect let us regard a single ring of electrons rotating round a nucleus of charge Ne, and let us assmne that an a-particle is expelled from the nucleus in a direction perpendicular to the plane of the ring. The expulsion of the particle will obviously not produce any alteration in the angular momentum of the electrons; and if the velocity of the a-particle is small compared with the velocity of "the electrons-as it will be if we consider inner rings of an atom of high atomic weight--the ring during the expulsion will expand continuously, and after the expulsion will take the position claimed by the theory ibr a stable ring rotating round a nucleus of charge (N--2)e. The consideration of this simple case strongly indicates that the expulsion of an a-particle will not have a lasting effect on the stability of the internal rings of electrons in the residual atom. The question of the origin of/9-particles may also be considered ti'om another point of view, based on a consideration of the ehemie~d and physical properties of the radioactive substances. As is well known, several of these substances have very similar chemical properties and have hitherto resisted every attempt to separate them by chemical means. There is also some evidence that the substances in question show the same line-speetrnm*. I~ has been suggested by several writers that tile substances are different only in radio-active properties and atomic weight but identical in all other physical and chemical respects. According to the theory, this would mean that the charge on the nucleus, as well as the configuration of the surrounding electrons, was identical in some of the elements, the only difference being the mass and the internal constitution of the nucleus. From the considerations of w 4 this assumption is ah'eady strongly suggested by the fact tlmt the number of radioactive substances is greater than the nmnber of places at our disposal in the periodic system. If, however, the assumption is right, the fact that two apparently identiefil elements emit /?-particles of different velocities, shows that the fl-rays as well as the a-rays have their origin in the nucleus. This view of the origin of a-and/~-partieles explains very simply the way in which the change in the chemical properties of the radioactive substances is connected with the Mr. R. V. Southwell on the nature of the particles emitted. The results of experiments are expressed in the two rules * :--1. Whenever an a-particle is expelled the group in the periodic system to which the resultant product belongs is two units less than that to which the parent body belongs. 2. Whenever a /3-particle is expelled the group of the resultant body is 1 unit greater than tha~ of the parent. As will be seen this is exactly what is to be expected according to the considerations of w 4. In escat)in_o-from the nucleus, the/~-rays may be expected to collide witch the bound electrons in the inner rings. This will give rise to an emission of a characteristic radiation of the same type as the characteristic RSntgen radiation emitted from elements Of lower atomic weight by impact of cathoderays. The assmnption that the emission of v-rays is due to collisions of fl-rays with bound electrons is proposed by Rutherford I" in order to account for the nmnerSus groups of homogeneous B-rays expelled from certain radioactive substances. In the present paper it has been attempted to show that the application of Planck's theory of radiation to Rutherford's atom-model through the introduction of the hypothesis of the universal constancy of the angular momentum of the bound electrons, leads to results which seem to be in agreement with experiments. In a later paper the theory will be applied to systems containing more than one nucleus. XXXVlII. On the Collapse of Tubes by External Pressure. --II. By R. V. SOUTnW~LL, 2B, A., Fellow of Trinity College, Cambridge ~. I N the issue of this Magazine for ~Iay 1913 w I have dealt with the effects of circular ends upon the resistance of tubes to collapse by external pressure, and in a somewhat fuller discussion oI the same problem ]] I have shown that my results are well supported by experiment, so far as the See A. S. Russell, Chem. News, cvii. p. 49 (1913) ; G. v. Hevesy, -phys. Zeitschr. xiv. p. 49 (1913) ; K. l?ajans, Phys. Zeitschr. xiv. pp. 1il & 136 (1913 ) : Ferh. d. deutsch. _Phys. Ges. xv. p. '240 (1913) ; 1 ~. Soddy, Chem. News, evil p. 97 (1913) . XXXVII. On the Constitution of Atoms and Molecules. By N. BOAR, Dr. phil. Copenhagen * PART II.--SYsTEMS CONTAINING ONLY A SINGLE NUCLEUS ~'.
w 2 .From 2 Configuration and Stability of the Systems.Let us consider an electron of charge e and mass m which moyes in a circular orbit of radius a with a velocity v small compared with the velocity of light. Let us denote the e 2 radial force acting on the electrons bY a: F; F will in general be dependent on a. The condition of dynamical equilibrium gives a --a 2Introducing the condition of universal constancy of the angular momentum of the electron, we have h
-~'~= h~ .......
o 20 v / ( a= + ~' = + ="-~a," cos a)" Putting in this expression z=0 and r =tan ~ a, and using the the radial force exerted on an electron in a point in the plane of the ring bU Ee ~-~;: = ~,~ q(~),
of the functions Q(~) and R(a) is given on p. 485. Next consider a system consisting of a number of concentric Downloaded by [Duke University Libraries] at 11:53 30 July 2012 rings of electrons which rotate in the same plane round a nucleus of charge ~qe. Let the radii of'the rings be al, as,...., and the number of' electrons on the different rings nl, 112, * ~ . * :Putting a~ = tan: (at,,), we get for the radial force acting as e 2
ratio between the radii of the rings tan e (~,.,,)= ~ . Tile values of Q(~) show that unless the ratio of the radii of the rings is nearly unity the offeet of outer rings on the dimensions of inner rings is very small, and that the corresponding effect of inner rings on out~er is Downloaded by [Duke University Libraries] at 11:53 30 July 2012
~' 2 are entire numbers, introducing the value~ for e, m, and h used on p. 479, we get for the factor before the bracket 3"1.1015.; the value observed for the constant in the Bahner spectrum is 3"290.10 ~. * This value is that calculated in the first part of the paper. Using the values e-~4"78. ]0 -l~ (see R. A. Millikan, Brit. Assoc. l~ep. ]912, p. 410),, e =5"31.1017 (see P.Gmelin~ .Ann. d. -phys. xxvili, p. 1086 (1909) and A. H. Bucherer, Ann. d. -phys. xxxvii, p. 697 (1912)), and :h -----7'27, 1016:(calculated by Planck's theory from the experiments of E. Warburg, G.Leith~user, E. Hupka, and C. ${iiller, Ann. d..Phys, xl. p. 611 (1913)) 2rr2e4m 1015 . we get ~ =3'26. in very close agreement with observations. Downloaded by [Duke University Libraries] at 11:53 30 July 2012 Dr. N. Bohr o~ the Constitution
Dr. N. Bohr on tl~e Constitutionremarkable homogeneity of the characteristic 1R(in~gei~ radiation--indicated by experiments on absorption of the rays, as well as by the interference observed in recent experiments on diffraction of RSntgen rays in crystals--is in agreement with the main assmnption used in Part I. (see p. 7) in considering the emission of line-spectra, viz. that the radiation emitted during the passing of the systems betwee~ different stationary states is homogeneous.
,~, o--2)n, m ; n, sn' -P,~o, --Pro m" 1 ............ table gives the values of s~ and p,,o--p .... from n=l to n=16. n, ~, P0 0 9 3"328 13'14 2 ............ 0"25 0"25 10 3"863 18"13 3 ............ 0'577 0'58 11 4'416 23'60 4 ............ 0"957 1-41 12 4"984 30"80 5 ............ 1"377 2"43 13 5"565 38"57 6 ............ 1"828 4"25 14 6"159 48"38 7 ............ 2"305 6"35 15 6"764 58"83 8 ............ 2"805 9"56 16 7"379 71"65
Communicar by Prof. E. Rutherford, F.R.S. t Part I. was published in Phil. Mug. xxvi. p. 1 (1913). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
Downloaded by [Duke University Libraries] at 11:53 30 July 2012
e 9 that ~ G,Sz is equal to the component perpendicular to the plane of the ring of the force--due to the nucleus and the electrons in the other rings--acting on one of the electrons if it has received a small displacement 8z. Using the same notation as above, we get G~= N--Zn,R(at~, ~). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
J. J.Thomson, Phil. Mat. xxiv. p. 218 (1912). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
* See J.Franck, Verb. d. Deutseh. _Phys. Ges. xii. p. 613 (1910). J. "W. Nicholson~ Month. Not. Roy. Astr.See. lxxiii, p. 382 (1913). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
(2, 1) a1=0"362 ao to1= 7"65 r o a~=1"182 a, to2=0"716 We W= 16"02 We.This configuration may be considered as highly probable also from a dynamical point of view. The deviation of the outermost electron from a circular orbit will be very small, partly on account of the great values of the ratio l~etween the radii, and of the ratio between the fl'equencies of the orbits of the inner and outer electrons, partly also on account of the symmetrical arrangement of the inner electrons.Accordingly, it appears probable that the three electrons Downloaded by [Duke University Libraries] at 11:53 30 July 2012
(4) a=0"329 ao to----9"26 too W= 37"04 We, Downloaded by [Duke University Libraries] at 11:53 30 July 2012
L Downloaded by [Duke University Libraries] at 11:53
July 2012
R. Whiddin~on, Prec. Roy. Soc. A. lxxxv, p. 323 (1911). 2 Comp. E.Rutherford, Phil. Mag. xxiv. p. 453 (1912). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
See A. S. Russell and R. Rossi, Prec. Roy. Soc. A. lxxxvii. I ). 478 ' (1912). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
t E.Rutherford, Phil. Mag. xxiv. pp. 453 & 893 (1912). $ Communicated by the Author.Vol. xxv. pp. 687-698. ~" On the General Theory of Elastic Stability," Phil. Trans.Roy. Soc. A. vol. ccxiii, pp. 187-244 (1913). Downloaded by [Duke University Libraries] at 11:53 30 July 2012
|
10.32388/01t7dq
|
cc-by
| null | null |
openalex
|
We are particularly interested in this paper because it explores a surprising finding in the alcohol-cognition literature we first stumbled on five years ago. The effect is an alcohol performance impairment that occurs only when task demands are relatively low. When the task is manipulated such that its cognitive load is increased, alcohol performance is equivalent to that of sober controls. We have since observed this exact form of interaction in two different contexts, during an attentional reaction time (RT) task (Bayless & Harvey, 2017) and in an inattentional blindness task (Harvey, Hyams & Bayless, 2018) . We have also revealed a closely related phenomenon in a test of number judgment (see Harvey & Seedhouse, 2021) . For alcohol to impair performance of an easy rather than more difficult version of a task is puzzling. We were therefore intrigued to discover Stock and colleagues had observed this effect in the context of response inhibition -the ability to withhold prepotent yet task inappropriate responses (Stock et al., 2014(Stock et al., , 2016)). In their most recent study (the focus of this review) Stock et al. ( 2022 ) investigated the boundary conditions under which acute alcohol intoxication increases impulsive responses in a test of motor inhibition known as the Go/No Go task.Participants were presented with a series of target stimuli (the items 5G7R) in either their mirrored or (standard) nonmirrored form. For high-demand trials (Block 1) they had to make a (button press) response only on non-mirrored "Go" trials (which occurred 70% of the time), with a left-hand button for letter stimuli and a right-hand button for number stimuli. For remaining (mirrored) "No Go" trials this habituated motor response had to be withheld. For low-demand trials (Block 2), they had to respond to letters (Go trials) but not numbers (No-Go trials), using a left-hand button press for mirrored letters and a right-hand button press for standard (non-mirrored) letters. Participants were tested under sober then alcohol intoxicated conditions. Task difficulty was manipulated by rotating each stimulus set such that it imposed either a low (30 o ), medium (90 o ) or high (150 o ) working memory load. A counterintuitive pattern of results emerged with alcohol reducing inhibition of "No Go" responses significantly more often for trials imposing the lowest (Block 2, 30 o ) rather than highest (Block 1, 150 o ) mental workload. We interpreted our alcohol-task difficulty interactions within the framework of alcohol myopia theory -the idea that alcohol depletes cognitive resources such that the focus of attention is restricted to only the most important or immediate stimuli (Steele & Josephs, 1990) . Our simple suggestion was that alcohol impairs performance under low but not high working memory demands because high load tasks are so challenging they leave little spare attentional capacity for the drug to deplete (Harvey et al., 2018; Harvey & Seedhouse, 2021) . Stock et al. (2022) , however, offer a more nuanced possibility. They suggest that motor responses to their easier Go trials are largely automated, thus demanding stronger inhibitions for successful No-Go trials -a cognitive control process which alcohol presumably weakens. For the harder task, a degree of complex cognitive control is needed even for Go trial responses, which the authors suggest is sufficient to disrupt automated responding itself thus negating the effects of alcohol. While developed to explain inhibitory task performance, this theory generalises to varying levels of cognitive control across other tasks (cf. Bayless & Harvey, 2017; Harvey et al., 2018) . In testing it, Stock et al. (2022) use EEG data to examine the neurophysiology of response inhibition under alcohol for low and high working memory demand versions of the Go/No Go task. They focus on event-related alpha and theta band activity -theta oscillations due to their association with cognitive control, inhibitory processes and WM; and alpha oscillations due to their association with WM and attentional control mechanisms. The authors suggest alcohol related effects on inhibitory processes are mediated by prefrontal cortical networks associated with WM and inhibitory processes, comprising the superior and middle frontal gyrus, and the inferior frontal gyrus. With the timing of these effects being either during the actual process of inhibitory control (up to 500ms after No Go stimulus presentation), or during the interval before the subsequent stimulus, reflecting alcohol myopia driven impairments to the anticipatory filtering (gating) of attention. Under low load conditions participants showed less theta desynchronisation when intoxicated than when sober (localised to the temporal cortex), indicating poorer attentional control under alcohol. Under high load conditions, however, less theta synchronisation (localised to central sites) was recorded when participants were intoxicated than when sober. This indicates increased attentional control under alcohol, consistent with the behavioural data. As expected, the authors also found the strongest source-level theta synchronisation under alcohol during low load trials, reflecting poorer inhibitory processing under these conditions. Plus, more synchronised task-related alpha band activity under alcohol for low load trials, suggesting less attentional gating under these conditions (localised to temporal and inferior frontal cortices). The problem with this account, however, is that it hinges on the assumption that low demand Go responses are predominantly automated, yet no behavioural or neural evidence is presented to support this claim. The analysis in the main paper focuses on false alarm rates and the underlying neural processing for inhibitory No-Go trials only. But Go trial measures are also needed to evaluate the impact alcohol had on these. It is possible, for example, that some participants sought to avoid errors of commission by adopting a more conservative and therefore effortful Go response strategy, particularly with under demanding task conditions. We note the authors' supplementary Go trial analysis, which indicates that alcohol had a significant effect on Go trials for both trial blocks. But data in this are collapsed across easy (30 o ) and hard (150 o ) task conditions. What is needed is a signal detection theory (SDT) analysis incorporating both Go and No-Go performance within the same inferential test. Specifically, comparing rates of Go trial hits, Go trial misses, No Go false alarms and correct No Go rejections (for details see, Young, Sutherland & McCoy, 2017) . This approach, along with a complementary analysis of reaction time (RT) data would provide a more complete understanding of alcohol effects and potential compensation effects across variations in task complexity. Further limitations to the study are outlined below. Two participants were excluded from the behavioural analysis for having Go trial scores substantially lower than the mean, though these participants were not excluded from the EEG analysis. This seems an odd decision so clarification on it would be helpful. A potentially more significant omission is the exclusion of the medium task difficulty data (trials at 90 o rotation) for which, in our view, no convincing reason is given. Responses to these trials should be incorporated into the main analysis, ideally using the SDT approach described above. Localisation of EEG signatures indicated some unexpected sources. The neural substrates for response inhibition and anticipatory gating are thought to be mostly in the prefrontal cortices, however, key differences between the alcohol and sober conditions were localised to temporal sites. The authors suggest that, in sequential tasks, aspects of response inhibition processing occur after the inhibitory act, which may therefore relate to bottom-up attentional processing common to occipital sites. However, a more elaborate discussion of this unexpected localisation of activity is warranted. EEG analyses are conducted on correct responses only, however, behavioural data are based on false-alarm rates, i.e., incorrect responses. In other words, the behavioural focus of alcohol's effect is on inhibition failures, while the neural focus is on inhibition success. It is not clear why this is the case. Were correct response trials emphasised to help identify the subsequent preparatory responses anticipated (e.g., attentional gating, etc.)? Alpha and theta band differences were found within a 500-1500ms time window, a latency corresponding to aspects of cognitive rather than motor processing, the latter being captured earlier (within 600ms of stimulus onset). The authors suggest that effects in later time windows may represent preparatory processes (in advance of subsequent trials), such as proactive control. This is an interesting idea that might have been addressed more specifically by analysing the time window time-locked to before the stimulus onset. For comparison, it may also be more reliable to run such an analysis on incorrect vs. correct trials. Finally, it would be of interest to mention whether there was an alcohol effect overall on the EEG signal, and if so what the characteristics of this were.
|
10.1186/1475-2875-11-27
|
cc-by
| null | null |
openalex
|
Background: Memory and learning are critical aspects of the ecology of insect vectors of human pathogens because of their potential effects on contacts between vectors and their hosts. Despite this epidemiological importance, there have been only a limited number of studies investigating associative learning in insect vector species and none on Anopheline mosquitoes. Methods: A simple behavioural assays was developed to study visual and olfactory associative learning in Anopheles gambiae, the main vector of malaria in Africa. Two contrasted membrane qualities or levels of blood palatability were used as reinforcing stimuli for bi-directional conditioning during blood feeding. Results: Under such experimental conditions An. gambiae females learned very rapidly to associate visual (chequered and white patterns) and olfactory cues (presence and absence of cheese or Citronella smell) with the reinforcing stimuli (bloodmeal quality) and remembered the association for up to three days. Associative learning significantly increased with the strength of the conditioning stimuli used. Importantly, learning sometimes occurred faster when a positive reinforcing stimulus (palatable blood) was associated with an innately preferred cue (such as a darker visual pattern). However, the use of too attractive a cue (e.g. Shropshire cheese smell) was counterproductive and decreased learning success.
Conclusions: The results address an important knowledge gap in mosquito ecology and emphasize the role of associative memory for An. gambiae's host finding and blood-feeding behaviour with important potential implications for vector control.Background As in many animals, memory and learning are fundamental aspects of the ecology of insect species. Visual place learning is critical for foraging and homing in most insects and is most conspicuously demonstrated in the social insects [5, 6] . Olfactory learning is equally important as it enables insects to optimize foraging and other activities in their local environment and has also been reported in several insect species . Understanding learning and memory bears particular importance for mosquitoes, sandflies, kissing bugs and other insects that transmit human diseases because their behaviour, particularly in relation to host finding and bloodmeal acquisition, may have critical implication for their vectorial capacity and thus the epidemiology of the diseases they transmit (reviewed in [10, 11] ). Despite its importance, there is a relative paucity of experimental studies focusing on memory in medicallyimportant insect species and much of the evidence stems from descriptive studies in which non-associative learning or habituation cannot be distinguished from associative learning . Habituation consists in the progressive decrease in the intensity of a pre-existing behavioural response to a stimulus that is neither particularly rewarding nor harmful. A classic example of habituation in insects is found in the Californian desert seed-harvesting ants Pheidole tucsonica and P. gilvescens, which were shown to decrease their level of aggression towards individuals from neighbouring colonies following repeated exposures to their odours . Habituation is thus a simple process enabling an insect's familiarization with its environment and an adjustment of its behavioural responses to it. In contrast, associative learning or conditioning occurs when an unpaired stimulus or cue (e.g. odour or visual pattern) becomes paired with a separate reinforcing or conditioning stimulus consisting in a benefit/reward or a cost/punishment leading to a behavioural change or the development of a new behavioural process altogether . For example, in bi-directional conditioning experiments with cockroaches, individuals presented with a peppermint smell associated with the reward of sugar water and vanilla odour associated with unpalatable quinine water, learned these associations and subsequently approached peppermint and avoided vanilla when exposed to these odours . Using the same positive (sugar water) and negative (quinine water) reinforcing stimuli, carpenter ants were trained to remember odours associated with them for up to three days and approached or avoided them accordingly . Evidence for learning and memory in mosquitoes stems from observations that in some species, males tend to exhibit swarm site fidelity and that in mark-release-recapture studies, females were sometimes recaptured in the location in which they were first captured (e.g. ). However, as critically reviewed by Alonso and Schuck , in most of these studies it is impossible to determine whether these few individuals exhibit site fidelity because of habituation, simply by chance, or because of characteristics of the environment itself. Indeed, because mosquitoes are expected to be found in parts of their habitat suitable for mating, oviposition, feeding, resting, etc., they are also more likely to be recaptured in those very same areas. Hence the non-random distribution of mosquitoes over habitats and their non-random exploitation of resources within habitats inevitably leads to some degree of apparent site-fidelity in recapture studies. In other studies, the design of the experiments did not allow for differentiating learning from possible innate preferences linked to different mosquito genetic backgrounds (e.g. [19, 20] ). Oviposition-site fidelity suggestive of olfactory memory has been reported in two studies in which chemicals with distinct smells were added to the larval rearing water of Culex quinquefasciatus and Aedes aegypti and the subsequent oviposition behaviour of adult females recorded. In both studies, females showed some degree of preference for laying eggs in the water they were reared in thereby supporting some process of preacquired knowledge of their former larval habitat characteristics. However, non-associative learning through familiarization or habituation is likely to be the process involved rather than associative learning . Since Alonso and Schuck-Palm's critical assessment of the evidence for memory in mosquitoes, associative learning has been specifically and directly tested in at least two experimental studies [22, 23] . Using glass pipettes mounted with a small odour-coated filter paper to present a reward in the form of sugar water or blood as reinforcing stimuli to males and female Cx quinquefasciatus, Tomberlin et al. were able to condition them into approaching the odour source and probe it even in the absence of the reward . Finally, larvae of Culex restuans were conditioned to associate predator odour (salamander odour) with the alarm cues of crushed conspecifics and subsequently displayed predator avoidance behaviour whenever exposed to the salamander odour . Here, a series of experiments aimed at describing the occurrence, extent and processes of associative memory and learning in Anopheles gambiae, the main vector of malaria in Africa, were performed. A simple assay was developed for bi-directional conditioning and testing of memory in groups of female individuals. Female mosquitoes were trained to avoid or be attracted to visual cues by associating these cues with a bloodmeal of low or high quality. Under such experimental conditions, An. gambiae learned very rapidly and remembered the association for up to three days. Importantly, associative learning and long-term memory could also be mediated via odour cues, but learning was affected by the type of odour used -i.e. if the odour was inherently attractive or not to female mosquitoes. The importance of these findings for An. gambiae vector capacity and their potential implications for vector control are discussed.
Methods
Standardized mosquito rearing A Mopti strain of Anopheles gambiae sensu stricto colonized in 2007 from field collections made in the region of N'Gabacoro Droit, in Mali, West Africa and maintained in F. Tripet's laboratory, Keele University was used for all experiments. The experiments themselves took place between 2007 and 2009 and thus involved many mosquito generations and many independent rearing episodes in order to obtain several replicates for each experiment. Mosquitoes were kept at 25C ± 1C and 70-80% relative humidity. In order to achieve homogeneity in phenotypic quality, all females used in behavioural studies were reared under standard conditions. Adult females were fed on horse blood using an artificial feeder (Hemotek membrane feeding system, Discovery workshops, UK). Eggs were laid two days post blood-feed and hatched within two days. A day later, newly emerged first instars were distributed into plastic trays (34 × 24 cm) at a density of 200 larvae per tray in 1 l of water. They were fed daily 80 mg of ground fish food (Tetra werk, Mulle, Germany) in order to yield adults of good size and phenotypic quality . Pupae were placed in a standard 5 l rearing cages for emergence and newly emerged male and female mosquitoes were kept together and with access to 5% glucose solution at all time. Standard rearing cages are made of 5 l white polypropylene buckets (~20.5 cm height × 20 cm diameter) with a sleeved side opening for introducing and removing mosquitoes and accessories, and the top covered with mosquito netting.
Bi-directional conditioning and short-term learning assay In all experiments, 40 two-to-six-day old females which had not yet been blood fed were collected from rearing cages and moved into a training/testing cage. The behavioural training/testing cages consisted in 5 l rearing cages similar to the usual rearing cages, but with a viewing window cut in its side covered by mesh. For all experiments, two Hemotek membrane-feeding system feeders were placed on top of a training/testing cage 5 cm apart. Learning was induced by associating a stimuli (visual, olfactory or other) with the action of landing on the feeder probing and feeding through the use of bi-directional conditioning through two contrasted reinforcing stimuli (reward/punishment) in the form of good and bad membrane qualities or palatable and unpalatable blood (see details below). The unconditioned stimuli and their associated feeder were placed on top of the testing cage and their position switched at a set time interval (2 min for short-term memory and 5 min for long-term memory experiments) for five intervals (Figure 1 ). Because An. gambiae females take ~10-20 min to feed to repletion, switching the position of the feeders interrupted their blood meal and females were thus forced to reposition themselves on the feeders several times in order to obtain a full bloodmeal. Whether the unconditioned stimuli became associated with the reinforcing reward/punishment through learning was recorded by counting the number of mosquitoes that landed on each feeder and initiated feeding using two click-counters.
Method validation experiments When performing conditioning/learning assays, in some instances the position of the feeders was switched at the 5th time interval, this time inverting the stimuli/feeder association. This enabled to regularly test whether mosquitoes effectively followed the conditioned stimuli through associative memory or if they used another source of information to directly track their preferred feeder. In addition, these 'method validation' experiments, demonstrated that females did not quickly move between feeders (from bad to good) thereby biasing our recording of their distribution at each time intervals.
Visual cues For studies on visual associative memory, two distinct visual stimuli or patterns were made using thick black and white paper. The patterns were shaped as circles that could fit around the two membrane feeders so that they would be clearly visible by mosquitoes resting on the cage walls or flying towards the feeders. Bi-directional visual conditioning was achieved by associating the contrasting colour patterns around the feeders with the action of landing and feeding via manipulation of the reward of the bloodmeal. At first, contrasting membrane qualities were used: sausage skin, which mosquitoes much prefer, was covered by a layer of Parafilm sealing film (Brand, Wertheim, Germany) and used on one feeder, and a double layer of Parafilm sealing film on the other. Two independent preliminary experiments on membrane preference showed that female An. gambiae simultaneously offered blood feeders covered with sausage skin, sausage skin covered with Parafilm, and Parafilm alone, significantly preferred sausage skin over the other two types of membranes and that sausage skin covered by Parafilm was significantly favored over Parafilm alone (Chisquare: P < 0.001 in both cases). Sausage skins were defrosted and soaked in water to expand them before use. The membranes was stretched over the blood holder and secured with a rubber collar. Any excess membrane from around the blood holder was then cut away. Both types of membranes were covered with an extra layer of Parafilm to make them appear visually identical. Using a micropipette, 1 ml of horse blood was distributed into each blood feeder. The Hemotek feeding system were set at 37C ± 1C for all experiments and the temperature of each feeder double-checked to ensure that they did not differ in temperature. In subsequent experiments, a much stronger differential reinforcing stimuli was created by drastically manipulating the quality of the bloodmeal contained in one feeder using salt. The blood in one feeder was made unpalatable (40% NaCl) whilst the blood in the other feeder was kept untouched (referred to as unsalted blood or good blood).
Olfactory cues For studies on olfactory associative memory, a 1 × 1 cm cube of Blue Shropshire cheese was placed in the bottom of a 2.5 × 5 cm black plastic container (film box). Cheese smell is known to attract mosquitoes because of similarities in odour compounds generated by the bacterial flora found on some cheeses and that living on human feet . Studying the interaction between odour attractants and learning is potentially relevant to the development of mosquito trapping devises (see discussion). Here, a film box with cheese was laid on its side next to the first membrane feeder and a similar but empty film box placed next to the second feeder so that they would look identical to mosquitoes resting on the cage walls or flying towards the feeders. Another set of experiments was made using Citronella essence, which is thought to act as a repellent against mosquitoes . Studying the interaction between odour repellents and learning is again potentially relevant for the efficacy of such compounds and their application to vector control (see discussion). A few drops of Citronella essence were dispensed on a small piece of cotton wool, which was again place within a film box (as above) laid on its side next to the first membrane feeder. A similar looking but empty film box was placed next to the second feeder. The Hemotek feeding system was set at 37C ± 1C for all experiments and the temperature of each feeder double-checked to ensure that it did not differ. Bi-directional olfactory conditioning was achieved by associating the location of the source of the smell with unpalatable (salted) or good (unsalted) blood.
Bi-directional conditioning and long-term learning assay Long-term learning/memory was studied using the exact same experimental designs as short-term learning except for the fact that previously trained mosquitoes were reassayed 24, 72 and 96 h after their first training/learning session. The initial distribution of mosquitoes on both feeders at the beginning of the training/learning session was then compared statistically to that at the onset of the retesting session.
Statistical analyses All statistical analyses were performed using the software JMP8.02 (SAS Institute, Inc). Pearson Chi-squares (likelihood ratios) were used to test the effect of the reinforcing stimulus position/side (e.g. position of salted blood versus non-salted blood feeders) on the distribution of probing and feeding females at a given time. Logistic regressions were conducted to study the combined effects of time (ordinal variable), the reinforcing stimulus position/side (e. g. position of salted blood versus non-salted blood feeders), the association between the reinforcing stimulus position and the visual or olfactory cue (e.g. salted blood with chequered colour pattern and non-salted blood with white pattern or reverse), and the interaction between these two factors on the number of females on the feeders (dependent variable). Logistic regressions were also conducted separately at given time intervals in order to compare the relative significance of each variable at each interval. The direct effect of replicate (variation in total number of mosquitoes feeding in a given experiment) and its interactions were removed from models except when this significantly affected the resulting model. Similarly all non-significant interactions between variables were manually removed from models using a stepwise procedure. Long-term memory was simply tested by comparing the number of mosquitoes recorded as probing or feeding on a feeder (dependent variable) at the beginning of the first training/learning session (first time interval) versus the beginning of the next session 24, 72 or 96 h later using logistic regression. The effects of the association between cues and the reinforcing stimuli were also included in these models.
Results
Associative visual memory Short-term memory Anopheles gambiae females quickly learned to recognize the chequered and white patterns circling the feeders and to associate that cue with the bi-directional reinforcing stimuli, high and low membrane quality (respectively sausage skin and Parafilm). The combined results of two replicates (chequered pattern with sausage skin and reverse) show that there was no significant difference in the numbers of mosquitoes feeding on each feeder 2 and 4 min after initiating the session, but from 6-10 min, there is a clear and significant preference for the feeder with the sausage skin membrane over that with a Parafilm membrane (Table 1 ).
Effect of reinforcing stimulus strength The same experiment conducted with the stronger bidirectional reinforcing stimuli of non-salted and salted blood led to similar results but females showed a significant preference for the feeder with non-salted blood already 4 min after the start of the session (Table 2 ). Comparing the relatively mild bi-directional reinforcing stimuli of the manipulation of membrane quality versus the drastic manipulation of blood palatability using logistic regression, revealed that the strength of the reinforcing stimuli significantly affected learning rate as evidenced by the strong significant interaction between reinforcing stimulus strength and the variable 'time' on the proportion of females feeding on each feeder (Logistic regression: n = 362, time: c 2 = 54.7, df= 4, P < 0.001; reinforcing stimuli strength: c 2 = 0.11, df= 1, p < 0.745, interaction: c 2 = 13.94, df= 4, P= 0.008) (Figure 2 ).
Effect of innate unpaired stimuli preference Adult female mosquitoes may innately prefer one unpaired stimulus or cue (here colour pattern) over another and, if that pattern is associated with the positive reinforcing stimulus (good feeder) during conditioning, this could affect learning rate. The potential effect of innate cue preference was explored by adding this as a variable 'association' in a logistic regression that included additional replication of the previous experiments (three replicates with association in both directions). Similar to what was found previously, on average females learned to follow the visual stimulus associated with non-salted blood from as early as the second interval (4 min) of the training/testing session to its end (10 min)(Chi-square: P < 0.001 in all cases). The direction of the association between colour patterns and reinforcing stimuli had a significant impact on learning. Overall, females preferred the association between chequered colour and non-salted blood than the reverse feeder/colour pattern association throughout the training session as evidenced by a significant 'association' variable in the model (Logistic regression: n = 386, time: c 2 = 24.8, df= 3, P < 0.001; association: c 2 = 8.42, df= 1, p= 0.004, interaction ns). Note that the data from time = 10 min was omitted in this analysis as a few late participating mosquitoes led to an artifactual low value for feeder preference (Figure 3 ).
Long-term memory Whether trained females remembered the association between a visual pattern and the unsalted blood for long periods of time after conditioning was tested by comparing their initial feeder preference at the beginning of the first training/testing session (untrained females) with their initial feeder preference in a second session (trained < 0.001 †The change in number of females on two blood feeders with contrasting bloodmeal quality (salted and normal blood) used as reinforcing stimuli associated with chequered and white visual patterns for bi-directional conditioning. The position of each feeder was alternated every 2 min (see methods for details). Significant results are in bold females) conducted 24, 72 and 96 h later (three independent experiments replicated three times in both directions for each re-testing intervals). Females significantly preferred the feeder with non-salted blood 24 h after the training session (Chi-square likelihood ratio: n= 105, c 2 = 4.49, df = 1, P= 0.034); their preference was very strong after 72 h (n= 119, c 2 = 13.25, p < 0.001), but disappeared after 96 h (n= 111, c 2 = 0.23, P= 0.636) (Figure 4 ). Note that the association between colour pattern and salted versus non-salted feeder was not significant at any of those time intervals (Logistic regression: P > 0.224 in all cases).
Method validation experiments Six replicates of method validation experiments (each involving both directions of the association between cue and reinforcing stimuli) in which the association of cue and conditioning stimuli was inverted in the 5th time interval were run in the course the study to verify that female mosquitoes effectively followed the cue associated with the preferred feeder because of learning. In all such experiments females were fooled into probing/ feeding onto the disliked feeder thereby proving that the conditioning/training sessions resulted in effective learning (Combined Chi-square: P < 0.001) (Table 3 ).
Associative olfactory memory
Short-term olfactory memory Anopheles. gambiae females also learned to recognize smells and to associate their intensity with the bi-directional reinforcing stimuli. In a first experiment, the smell of Blue Shropshire cheese was associated with the bi-directional reinforcing stimuli of salted or not salted blood. The combined results of two replicates (strong Figure 2 Effect of the strength of the reinforcing stimulus on learning rate in Anopheles gambiae females. The mean proportion (± 95%CIs) of females probing and feeding on the preferred feeder associated with the positive reinforcement is shown for two sets of experiments. In the first experiment (white circles) the positive and negative reinforcing stimuli were the preferred sausage-skin membrane or the disliked Parafilm membrane; in the second (black circles), unpalatable salted blood and non-salted blood was used (see text and Table 1 , 2 for details). Figure 3 Effect of innate preference in unpaired visual stimulus on learning rate in Anopheles gambiae females. The mean proportion (± 95%CIs) of females probing and feeding on the preferred feeder associated with the positive reinforcement is shown for two sets of replicates of the same experiment. The positive reinforcing stimulus of non-salted blood was associated with the innately preferred chequered visual pattern in the first set (white circles) and with the lessfavoured white pattern in the second (black circles). Figure 4 Long-term memory retention in Anopheles gambiae females previously conditioned to associate visual cues with non-salted or salted blood. The mean proportion (± 95%CIs) of females probing and feeding on the preferred feeder associated with the positive reinforcement (non-salted blood) is shown at the beginning of the training experiment (white circles) when the stimulus is still unpaired, and at the beginning of the re-testing session. Independent experiments tested memory retention after 24 h, 72 h, and 96 h (see test for details). Significance levels are P < 0.05 *, P < 0.005 , P < 0.001 *, P > 0.05 ns. cheese smell with salted blood and reverse) show that there was no significant difference in the numbers of mosquitoes feeding on each feeder 2, 4 and 6 min after initiating the session, but at 8 (one-way test) and 10 min, there was a significant preference for the feeder with non-salted blood over the salted one (Table 4 ). The direction of the association between smell and salted/non salted blood significantly affected the distribution of females on the feeders, with the cheese/nonsalted blood attracting more females, and this was true for all time intervals (4, 6, 8 and 10 min) except the first one (Logistic regression: P < 0.001 in all cases).
Effect of different unpaired stimuli In a second experiment, the smell of Citronella was associated with salted/non-salted blood. The combined results of two replicates (Citronella smell with normal blood and reverse) show that female mosquitoes learned to associate the Citronella smell and blood quality already 4 min after initiating the session and stayed choosey until the end of the session (Table 5 ). The direction of the association between Citronella smell and salted/non salted blood did not significantly affect the distribution of females on the feeders but there was a significant effect of time, consistent with the results of Table 5 (Logistic regression: n= 327, time: c 2 = 18.2, df= 4, p= 0.001, association: c 2 = 0.32, df= 1, p= 0.573, interaction ns). Comparing the effect of different innate unpaired cue -i.e. the attractive cheese smell versus Citronella -using logistic regression, revealed significantly better learning when using Citronella rather than cheese (Logistic regression: n = 557, time: c 2 = 16.9, df= 4, p= 0.002; stimulus type: c 2 = 12.2, df= 1, p < 0.001, replicate: c 2 = 31.08, df= 6, p < 0.001) (Figure 5 ).
Long-term olfactory memory Whether trained females remembered the association between the attractive Blue Shropshire cheese smell and salted or non-salted blood 72 h after the initial training/ learning session was tested by conducting a second session and then comparing the female initial distribution on the feeders at 2 min in the two sessions (two independent experiments replicated two times in each direction). No statistical difference between the two sessions was found and the main determinant of female distribution on the feeders was the association of cheese smell with the feeder with non-salted blood (Logistic regression: *Here the association between the visual stimulus and blood quality was inverted †The change in number of females on two blood feeders with contrasting bloodmeal quality (salted and normal blood) associated, in this instance, with chequered and white visual patterns for bi-directional conditioning was recorded. The position of each feeder was alternated every 2 min; but at time 10 min (in bold) the association between visual cue and feeder quality was inverted in order to verify that females effectively followed the cue and did not simply distinguish feeders. Females were thus fooled into probing/feeding on salted blood. Significant results are in bold 0.005 †The change in number of females on two blood feeders with contrasting bloodmeal quality (salted and normal blood) used as reinforcing stimuli associated with and without Shropshire cheese smell for bi-directional conditioning was recorded. The position of each feeder was alternated every 2 min (see methods for details). Significant results are in bold (two-way test) and labelled with an asterisk (one-way test) session: n= 123, session: c 2 = 0.09, df= 1, P= 0.768, association: c 2 = 8.40, df= 1, p= 0.004, interaction ns). The same analyses, was used to test for long-term memorization of the association between the disliked Citronella stimulus and salted/non-salted blood. Across the two sessions, females preferred the feeder with nonsalted blood but this time that preference was independent of the direction of the association. Females significantly remembered the association after 72 h as evidenced by the significant effect of session on the number of females on each feeder (Logistic regression: n= 192, session: c 2 = 4.77, df= 1, P= 0.029, association: c 2 = 0.01, df= 1, p= 0.970, interaction ns).
Discussion
Visual memory This study reports strong evidence of short-term memory in response to conditioning with visual (chequered and white patterns) cues. To our knowledge, this is the first study that experimentally conditioned mosquitoes into associating visual patterns with a reinforcing stimulus, here bloodmeal quality, thereby demonstrating the occurrence of visual memory. Interestingly and predictably, learning rate depended on the strength of the bi-directional positive and negative reinforcing stimuli. In a first series of experiments, the preferred sausage skin membrane and the less liked Parafilm were used as reinforcing stimuli, which resulted in learning but at a slow rate. In several instances it took more than the five intervals (10 min) to train all mosquitoes, leaving more opportunities to have spurious results in the last interval generated by a few late-feeding females. This slower learning rate is not surprising because the negative reinforcing stimulus of the Parafilm still equated to a successful bloodmeal, hence a profitable experience for feeding females. Adding salt to the blood allowed for a truly negative reinforcing stimulus as feeding was prevented altogether due to bloodmeal unpalatability. This resulted in a much stronger differential conditioning and faster learning. Indeed the distribution of females on both feeders suggested that a single attempt at feeding on the salted blood may have led to effective conditioning. The use of salt here had the same effect as the quinine used in previous studies on other insect species (e.g. [7, 14, 22] ). Another factor that significantly affected learning was the 'direction' of the association between unpaired and reinforcing. Mosquitoes prefer darker locations where they are inconspicuous. As a result, females had an initial innate preference for the feeder with chequered pattern that made them feel safer while feeding, and consequently learned to associate that pattern with the positive reinforcing stimulus of a salt-free bloodmeal significantly faster than when the patterns was associated with salted blood. The mean proportion (± 95%CIs) of females probing and feeding on the preferred feeder associated with the positive reinforcement stimulus of non-salted blood is shown for two sets of experiments. In the first experiment (white circles) the olfactory cue of Blue Shropshire cheese smell was associated with one of the feeder and the other feeder was kept odourless or vice-versa; in the second (black circles), Citronella smell was used in a similar way (see methods for details). That female mosquitoes effectively learned to follow the cues associated with their preferred bloodfeeder was tested several times in the course of this study and simply demonstrated by changing the association between the cue under study and the reinforcing stimuli at the last time interval of an experiment. It is noteworthy that nearly all females were successfully fooled into probing/ feeding on bad quality blood in such 'method validation experiments'. These results also enabled us to dismiss the possibility that females would actively sample both feeders before settling on the better tasting one within the time elapsed between mosquito counting on each feeder. An. gambiae females seldom change location once they start feeding on a feeder probably because changing biting site carries important costs to feeding females in nature. Bi-directional conditioning led to long-term visual memory with mosquito females remembering the colour pattern associated pattern for as long as 24 and 72 h but not 96 h. This duration is comparable to that found in a bidirectional conditioning experiments conducted on carpenter ants . It is worth pointing out that in honeybee studies that have examined those aspects in detail, longterm association retention usually depended on the number of conditioning trials , but also on stimulus strength . In preliminary experiments (results not shown), it was not found to be possible to generate visual memory retrieval when using the moderate differential stimuli of membrane quality (see above), however experiments using salted blood led to successful visual memory retention. One potential biological explanation for the three-day retention duration could be that spatial memory associated with a bloodmeal is linked to the context of host finding and feeding in mosquito females, hence depends on their gonotrophic cycle which takes two to two-and-half days in the wild [29, 30] . Spatial memory would help females return to a feeding location or to a host on which they fed successfully in their previous gonotrophic cycle, as suggested by some studies . This would thus minimize the costs associated with host finding and maximize foraging efficiency.
Olfactory memory Importantly, similar results using olfactory cues were found, albeit those results strongly depended on the type of odour used. Females learned to associate both Blue Shropshire cheese and Citronella smells. However, there was such a strong innate attraction for the cheese smell that learning was significantly hindered when the cheese smell was associated with salted blood. When Citronella was used, the direction of the association between presence/absence of odour and salted/non-salted blood did not affect learning significantly. This may seem to contradict evidence showing that Citronella is an effective mosquito repellent [26, 32] . However formulation and the mode of application are important for effectiveness; and here, because females did not have to be in direct contact with Citronella essence, this may have decreased its repellency. Importantly, learning was achieved at an overall faster rate with Citronella than with cheese because of the similarity in learning efficiency regardless of the direction of the association between odour and reinforcing stimuli. This had drastic consequences for long-term olfactory memory retention, as significant memory retrieval could not be detected following the partial conditioning achieved with cheese odour but females remembered the associations with Citronella smell for 72 h. No attempt were made to prolong the training/conditioning session with cheese until all females learned to avoid the smell of cheese, but it is unlikely that this would have improved memory retention, such is their innate attraction to some cheese varieties . The fact that females could retain memory of an association between odour and a negative physiological experience such as probing/tasting very salty blood suggest that associative learning could potentially play a role in behavioural avoidance of pesticides. If host-seeking females encounter sub-lethal doses of insecticide whilst seeking hosts, there is certainly no reason why they should not learn to associate their smell and ill effects and subsequently avoid these pesticides when they smell them. Thus the exact role and importance of learning and memory in insecticide excito-repellency remain to be investigated. A role for learning and memory could generally also be expected in devices or vector control strategies that would make use of attractants (visual or olfactory) in combination with a vector killing system that does not provide full lethality, such as sticky traps or exposure to partially lethal pathogens.
Methodological limitations The bi-directional dual-feeder conditioning experimental cage set-up developed for these studies enabled to condition groups of female mosquitoes. This was a major improvement over the individual-based approach previously used in studies of associative memory in adult mosquitoes , cockroaches , or ants . This novel approach enabled to quickly build-up sample sizes, compare experimental groups or replicates; hence explore relatively quickly more complex processes of associative memory. The downside of such approach is the lack of real control over which females were actually exposed to the contrasted bloodmeal quality/visual or olfactory cues association because they cannot all be forced to feed and participate in the experiments. Thus varying amounts of variation in the analyses were attributable to varying number of females that were not hungry at the time of the conditioning experiments. Other females initiated their feed after a certain delay, hence joined the experiment near its end, thereby creating spurious background noise (e.g. Figure 3 at 10 min). Mosquito rearing was thus critical in ensuring that larvae were reared under standard conditions and that they resulted in adults of homogenous quality and behaviour. This helped ensure that a majority of females fed at the start of the training/ testing session, which is critical for most experiments but particularly for long-term memory ones in which females were re-tested in the following days.
Conclusions The results show that Anopheles gambiae females were capable of associating visual and olfactory stimuli with a positive or negative reinforcing stimulus after only one or two conditioning exposures to that association. This resulted in short-term but also long-term memory with a maximum retention time of 72 h. Learning depended on the strength of the reinforcing stimulus used and, when innate preferences to a visual or olfactory cue were involved, on the direction of the association between the two visual or olfactory stimuli and the positive and negative reinforcing stimuli used for bi-directional conditioning. The possibility that host-seeking females would return to preferred memorized feeding locations and hosts confirms the potential importance of memory for mosquito optimal foraging and suggest that it may also enhance underlying heterogeneities in host exposure to bites. The generally understated importance of learning for mosquito vectorial capacity and the epidemiology of the diseases they transmit is particularly obvious in the light of its potential role in acquired behavioural avoidance to insecticides. Figure 1 1 Figure 1 Experimental set-up for visual conditioning training and testing experiments. Two blood feeders with either a white or chequered visual pattern around them (underside view depicted here) were placed on the top of a 5 l experimental training/testing cage. Their position was alternated every 2 or 5 min (see text for details) for five times and the number of females on each feeder recorded at each time interval.
Figure 5 5 Figure5Effect of different types of unpaired olfactory stimuli on learning rate in Anopheles gambiae females. The mean proportion (± 95%CIs) of females probing and feeding on the preferred feeder associated with the positive reinforcement stimulus of non-salted blood is shown for two sets of experiments. In the first experiment (white circles) the olfactory cue of Blue Shropshire cheese smell was associated with one of the feeder and the other feeder was kept odourless or vice-versa; in the second (black circles), Citronella smell was used in a similar way (see methods for details).
Table 1 1 Bi-directional conditioning experiment for short-term visual memory in Anopheles gambiae females using contrasted membrane qualities Both membrane types were covered by an extra layer of Parafilm to ensure similar appearancesThe change in number of females on two blood feeders with contrasting membrane qualities (sausage skin or Parafilm) used as reinforcing stimuli associated with chequered and white visual patterns for bi-directional conditioning was recorded. The position of each feeder was alternated every 2 min (see methods for details). Significant results are in bold Association Chequered/Sausage skin White/Sausage skin c 2 p-value Membrane Sausage skin † Parafilm † Sausage skin † Parafilm † - - Time (min) n n n n 2 17 15 10 9 0.17 0.674 4 18 10 11 7 3.17 0.075 6 17 8 13 4 7.70 0.006 8 15 4 12 4 10.89 0.001 10 18 1 14 3 24.79 < 0.001 †
Table 2 2 Bi-directional conditioning experiment for short-term visual memory in Anopheles gambiae females using contrasted blood qualities Association Chequered/Normal blood White/Normal blood c 2 p-value Blood Normal blood † Salted blood † Normal blood † Salted blood † - - Time (min) n n n n 2 9 7 8 6 0.5 0.465 4 11 4 10 2 8.8 0.003 6 16 1 10 0 28.9 < 0.001 8 19 0 15 0 47.1 < 0.001 10 18 0 16 0 47.1
Table 3 3 Example of method validation experiment using contrasted blood qualities Association Chequered/Normal blood White/Normal blood c 2 p-value Blood Normal blood † Salted blood † Normal blood † Salted blood † - - Time (min) n n n n 2 14 16 9 7 0.0 1.000 4 19 7 9 4 7.2 0.006 6 15 4 16 4 9.9 0.002 8 17 0 17 3 30.5 < 0.001 10* 0 16 1 20 42.1 < 0.001
Table 4 4 Bi-directional conditioning experiment for short-term olfactory memory to cheese smell in Anopheles gambiae females Association Cheese/Normal blood No cheese/Normal blood c 2 p-value Blood Normal blood † Salted blood † Normal blood † Salted blood † - - Time (min) n n n n 2 10 4.5 10.5 9.5 2.46 0.115 4 6.5 1.5 6 9 0.35 0.555 6 7 1 5.5 7 1.99 0.158 8 6 0.5 7 6.5 3.66 0.056* 10 6 0 6.5 4.5 7.84
Table 5 5 Bi-directional conditioning experiment for short-term olfactory memory to Citronella smell in Anopheles gambiae females †The change in number of females on two blood feeders with contrasting bloodmeal quality (salted and normal blood) used as reinforcing stimuli associated with and without Citronella smell for bi-directional conditioning. The position of each feeder was alternated every 2 min (see methods for details). Significant results are in bold Association Citronella/Normal blood No Citronella/Normal blood c 2 P-value Blood Normal blood † Salted blood † Normal blood † Salted blood † - - Time (min) n n n n 2 15 13 9 11 0.00 1.000 4 14 7.5 11 6 6.98 0.008 6 14 4.5 11 2.5 21.5 < 0.001 8 8 2 12 6.5 9.55 0.002 10 5.5 2 7.5 1.5 11.64 < 0.001
|
10.3389/fninf.2019.00027
|
cc-by
| null | null |
openalex
|
This work presents a pieces of Python code to rapidly simulate the spiking responses of large numbers of single cutaneous tactile afferents with millisecond precision. To simulate the spike responses of all the major types of cutaneous tactile afferents, we proposed an electromechanical circuit model, in which a two-channel filter was developed to characterize the mechanical selectivity of tactile receptors, and a spike synthesizer was designed to recreate the action potentials evoked in afferents. The parameters of this model were fitted using previous neurophysiological datasets. Several simulation examples were presented in this paper to reproduce action potentials, sensory adaptation, frequency characteristics and spiking timing for each afferent type. The results indicated that the simulated responses matched previous neurophysiological recordings well. The model allows for a real-time reproduction of the spiking responses of about 4,000 tactile units with a timing precision of <6 ms. The current work provides a valuable guidance to designing highly realistic tactile interfaces such as neuroprosthesis and haptic devicesINTRODUCTION Tactile receptors are sensory receptors that respond to mechanical pressure or distortion by producing action potentials (spikes) in their associated afferents (Zhu and Rozell, 2015) . Previous neurophysiological research has found that four types of tactile afferents in skin are responsible for tactile sensation (Vallbo and Hagbarth, 1968; Johnson, 2001) . Different types of tactile afferents respond to mechanical stimuli with different selectivity. Slowly adapting type 1 (SA1) afferents respond to sustained pressure and low-frequency vibrations and convey information about shape (Goodwin et al., 1997) . Rapidly adapting type 1 (RA1) afferents respond to stimuli that bump against the skin and convey information about motion across the skin (Johansson and Westling, 1987) . The RA1 and SA1 afferents account for spatial acuity in psychophysical tests (Johansson and Vallbo, 1979) . Rapidly adapting type 2 (RA2) afferents, also called Pacinian Corpuscle (PC) afferents, are very sensitive to high frequency vibrations and are thought to convey information about distal events such as texture roughness sensed through a tool (Yoshioka et al., 2007) . Slowly adapting type 2 (SA2) afferents are sensitive to skin stretch and may provide information about the hand conformation and posture during grasping and other hand movements (Collins et al., 2007) . The SA2 afferents have never been observed in neurophysiological studies of mechanoreceptors in the monkey hand (Johnson, 2001) . While the response properties of three major types of tactile afferents (SA1, RA1 and PC) have been extensively studied in neurophysiological recordings. Therefore, a model of reproducing responses of SA1, RA1 and PC afferents is feasible, and valuable to investigating tactile sensation. Generally a model of simulating cutaneous afferent response should at least consist of the receptor model to emulate the selectivity of each afferent type, and the spiking neuron model to generate action potentials in afferents. In current work we treat the tactile receptor and its associated afferents as a single tactile unit. A number of influential models have been developed to characterize how a spiking neuron produce action potentials. The most important and accurate of the early neural models is the Hodgkin-Huxley model (Hodgkin and Huxley, 1952) , which describes a spiking neuron by a coupled set of four ordinary differential equations (ODEs). The Hodgkin-Huxley model has inspired several more simplified models, such as the Morris-Lecar model, FitzHugh-Nagumo model and Izhikevich model, all of which have two coupled ODEs (Fitzhugh, 1961; Izhikevich, 2003; Tsumoto et al., 2006) . The integrate-and-fire (IF) model is another spiking neuron model that has computational simplicity, scalability and applicability to simulate SA1, RA1, and PC afferents (Bensmaia et al., 2008; Becker et al., 2009; Saal et al., 2017) . The previous work of spiking neuron has been fruitful. Besides in neuromorphic applications, some models of mechanotransduction have also been reported to characterize the tactile afferents by using above spiking neuron models (Spigler et al., 2012; Rongala et al., 2015; Nguyen et al., 2018; Osborn et al., 2018) . However without considering the filter characteristics of tactile receptor, the reproducing of selective responses (e.g., frequency and adaption) of the three major types of tactile afferents is not mentioned in these work. By combining a filter model of tactile receptor with IF neuron model, some researchers developed more comprehensive models that can replicate many response properties of SA1, RA1, and PC afferents and even the afferent population responses (Dong et al., 2013; Saal et al., 2017) . However, most previous spiking neuron models including the IF model fail to accurately recreate the neural spiking responses of tactile afferents, particularly for the PC afferents (Biswas et al., 2015) . Accurate characterization of the evoked action potentials in response to a stimulus is paramount to designing neuroprostheses with biological compatibility (Marmarelis, 1993; Bertaccini and Fanelli, 2009) . In tactile neural nerve system, the spike timing is important to encoding object properties including vibratory frequency, surface texture, and surface curvature (Saal et al., 2016) . Recent literature hints that the binary spike trains of the sensory receptors can be quite accurately modeled with an pulse frequency modulator (PFM) (Biswas et al., 2015) . The non-linear stochastic mechanotransduction (NSM) model consisting of a mechanic filter and an adaptive relaxation PFM allows for accurate reproduction of the spiking response of PC afferent and captures the shape of the action potential more accurately than the IF model (Biswas et al., 2015) . Hower the NSM model which has more than 10 coupled ODEs can only be implemented to recreate the responses of PC afferents, and is too complicated to allow real-time simulation of massive numbers of tactile units. The goal of current work was to propose an model that could accurately recreate most single afferent response properties observed in previous literature, but that was simple enough to allow for fast computation. Most previous models of recreating afferent response include a model of skin mechanics. However, previous studies have shown that incorporating implementing skin mechanics is not necessary to achieve high spiking precision for only reproducing responses of the tactile afferents located under stimuli sites (Saal et al., 2017) , and previous work was able to reproduce precise spiking patterns evoked by vibrating probes without implementing a skin mechanics model (Kim et al., 2010; Dong et al., 2013) . The model of skin mechanics can be seen as a model of computing the indentation distribution over the skin from the pressure distribution. There are already much experimental data about tactile afferent responses to skin indentation depth (not applied force) in neurophysiological literature [e.g., Muniak et al. (2007) ]. Under a certain area of surface indenting the skin, the caused indentation depth on stimulus site change linear with applied pressure according to elastic theory (Timoshenko and Goodier, 1970) . In order compare simulated responses with well-established response properties in previous neurophysiological literature, the current work only focus on simulating afferent responses of single unit to skin indentation, which is the basic work for recreating afferent population responses. A cutaneous tactile unit involves not only the filtering of the mechanical signal but also the modulation of the electrical signal (Biswas et al., 2015) . Inspired by previous filter models of tactile receptor and frequency modulator of generating spikes, we proposed an electromechanical circuit model consisting of a two-channel filter (TCF) to characterize the mechanical selectivity of a single tactile receptor and a spike synthesizer using frequency modulator to accurately recreate action potentials evoked in afferents. In the TCF model, the signal of skin indentation is selected by the twochannel filter, in which a low-pass filter (LPF) and band-pass filter (BPF) were designed to select the static and dynamic components of the response, respectively. The spike synthesizer was designed to convert the the output signal of TCF model into action potentials. The main contributions of this work are as follows: (1) it presents a electromechanical circuit model that allows a rapid simulation of thousands of SA1, RA1, or PC afferents in response to skin indentation; (2) it presents a spike synthesizer to rapidly generate action potentials with millsecond timing precsion, which may improves the biological compatibility for neural prostheses; (3) it presents a method to train the parameters of the TCF model using neurophysiological firing-rate datasets and yield spike timing precision of <6 ms.
METHODS The electromechanical model as illustrated in Figure 1A was developed to replicate the afferent response properties of single tactile unit. The whole model is comprised of the TCF model to characterize mechanoreceptive end-organ (the receptor) and other models (Transducer, Normalizer, and Spiking synthesizer) to characterize afferent fiber (the nerve).
Electromechanical Circuit Model A good way to investigate the response properties of single tactile unit is isolating from the afferent population (Muniak et al., 2007) . Previous neurophysiological experiments of single tactile unit has indicated that stimulus intensity is encoded in the firing rate (ips, impulses per second) of action potentials rather than its firing amplitude (Goodwin et al., 1997; Muniak et al., 2007) . In this work, we proposed the TCF model to quantitatively replicate the firing-rate response (v nf ) of a tactile unit to mechanical indentation, then constructed a spike synthesizer to convert the signal of v nf to corresponding to spikes. The main source code that implements the proposed model is depicted in Figure 1D , which are available in the link: https://github.com/ouyangqq/model_of_single_tactile_ unit/blob/master/Receptors.py. In this code, all the intermediate and I/O signals were defined as two-dimensional Numpy arrays (spatial * temporal) to support simulating afferent population responses in future. To speed up computation, all logistic and loop operations were converted into vector or matrix operations using Numpy library imported as "np" in the source code.
Model of Receptor Previous neurophysiological experiments indicated that each type of single tactile unit appears to mediate specific portions of the overall stimulus frequency characteristic (Bolanowski et al., 1988) . SA1 afferents are most sensitive between 0.3 and 3 Hz (Johnson et al., 2000) , RA1 afferents between 2 Hz and 50 Hz, and PC afferents between 30 and 500 Hz (Mountcastle et al., 1972) . These characteristics of neural threshold are close to characteristics of a filter to the mechanical stimulus (skin indentation). Thus, we designed a two-channel filter to select the mechanical stimulus in frequency range of sensitivity for each type of tactile units. The Laplace transfer function of the filter is given as below. H (s) = S m (S) x in (S) = K b1 S + K b2 S 2 + + K b n S n S + 2π fBL 2π fBH S + 2π fBH n+1 BPF + Ku 2π fL S + 2π fL LPF (1) Where K u and K b(1∼n) are weights of the low-pass filter (LPF) and the band-pass filter (BPF), respectively. n is the highest order of taking derivation to the input stimulus. f BL and f BH are the lower limit and upper limit cut-off frequencies of the BPF, respectively. f L is the cut-off frequency of the LPF. As illustrated in Figure 1A , the outputs of the two filters are added together. x in , s m in equation ( 1 ) is the input stimulus and the added signal of the two filters, respectively. For convenience of processing, the added signal was rectified to a positive signal. w is the negative contribution for rectifying. w = 0 for SA1 afferents, since they tend not to respond to the offset of stimuli (Mountcastle et al., 1966) ; w > 0 for RA1 and PC afferents. A s is the mechanotransduction coefficient. The transfer function H (S) was converted to a differential equation, which can be discreetly solved in loop iterations as shown in Figure 1D . In the TCF model, the LPF mainly cares about the static component of the input stimulus, while the BPF is only sensitive to changes of stimulus (the dynamic component). According to previous work, the SA1 afferent is not only sensitive to static but also dynamic stimuli (Johnson, 2001) . SA1 afferents respond approximately ten times more during dynamic stimulation than during static stimulation (Johnson et al., 2000) . K u was set to zero For the RA1 and PC afferents, since they adapt so quickly and tend not to respond during static stimulation (Johnson, 2001) . As illustrated in equation ( 1 ), the current model includes several inertia components (1/(S+2π f )), which causes different time delay between mechanical deformation on the skin surface and mechanical deformation of the receptor for each type of tactile units.
Model of Afferent Fiber The transducer in Figure 1A proportionately converts the mechanical signal to electric current (A s proportion coefficient of transducer), and drives the subsequent circuit to evoke spike trains in the ascending afferents. The tactile afferents tend not to fire until the stimulus amplitude exceeds a minimum amplitude (the lower threshold) (Vallbo and Johansson, 1984) , and the firing rate of tactile afferents do not increase when the stimulus amplitude exceeds a maximum amplitude (the upper threshold) (Freeman and Johnson, 1982) . To simulate this neural threshold property similar with a normalizer, a rectifying diode D 1 of lower limit, and a Zener diode D 2 of upper limit were added in the model. Turn-on voltage (V L ) of D 1 was set as the lower limit of the action potential relative to the resting state (Figure 1C ). For a typical neuron, the resting potential is around -70 millivolts (mV) and the threshold potential is around -55 mV. Thus, in the current study, V L was set to 15 mV. The breakdown voltage (V H ) of D 2 was set to 1,000 mV. The frequency response characteristics of the filter can obtained by substituting S = j2πf into equation ( 1 ). The frequency response characteristics of the neural threshold (um) is given as follows: T (f ) = V L A s |H j2πf | (2) Where j is the imaginary unit, f is the frequency. The lowest indentation threshold (T low ) of a tactile unit can be evaluated as following equation. T low =T f BL +f BH 2 = V L A s |H j2π( f BL +f BH 2 ) | (3) The spike synthesizer as illustrated in Figure 1B was developed to produce a biological neural spike with a firing rate that is proportional the signal of v nf . v nf is the output of the neural threshold model. The v nf was sampled (sampling period: T a ) with holding into v s , and the v s was then modulated into the frequency-adjustable triangular wave (v f ). The v f was then compared with a constant (0.5) to produce a pulse wave (v r ). The K f in Figure 1B was defined as the max firing-rate for each afferent tpye. f c (1/T c =K f v s ) is the frequency of carrier wave. The action potentials (v a ) were finally generated by superposing the base wave from rising edge of the pulse wave. The base wave was synthesized with a typical spike shape as shown in Figure 1C . T a is the period of a spike, which was set to 4 ms for each afferent type.
Model of Noise As shown in Figure 1A , the mechanical and neural noises are separately incorporated in current model. The reason behind these two separate provisions is that the non-linear signal processing in tactile receptor and afferent fibers alters the statistical and morphological properties of mechanical noise (x N ) and neural noise (v N ) differently (Muniak et al., 2007) . For simplicity the mechanical noise was considered as pseudo-Gaussian noise with 0.1 um of standard deviation and filtered with 1,000 Hz cut-off first-order low pass filter (Biswas et al., 2015) . Neural noise was modeled as an additive random noise associating with the action potential (v a see Figure 1C ). For simplicity the neural noise was considered as pseudo-Gaussian noise with SD = 10 mv and filtered with 1,000 Hz cut-off firstorder low pass filter.
Model Training The neurophysiological firing-rate datasets (recorded from rhesus macaques, see Figure 3 in Muniak et al., 2007) in response to sinusoidal vibrations, were used as the training data. Before training, the firing-rate data was normalized using a feature scaling method to bring all values into the range of 0 to 1. The normalized firing rate of this model in response to peak amplitude (x) of sinusoidal stimulus and frequency (f ) is illustrated in the following equation. h θ x,f =A s |H j2πf x | 1+w π (4) Using the above equation, the loss function of training the model was obtained as follows. J (θ ) = 1 2 m i=0 h θ x (i) ,f (i) -y (i) 2 (5) Where m is the size of the training dataset, y (i) is the target value of i th training set. θ = [K b1 , K b2 , . . . , K bn , K u , f BL , f BH , f L , A s , w] is the parameter array of the model. The goal of optimizing the parameters of the model is to find values of θ so that J (θ) reaches its minimum. There are three major parameters optimization methods: gradient descent (steepest descent), Newton's method and the quasi-Newton method. The quasi-Newton method is good enough to produce superlinear convergence to a global minimum, thus the improvement over the steepest descent is dramatic especially on difficult problems (Wright and Nocedal, 2006) . Since second derivatives are not required, the quasi-Newton method is more efficient than Newton's method (Wright and Nocedal, 2006) . We implemented the BFGS quasi-Newton method to train the parameters using Scipy.optimize library (https://github.com/ scipy/scipy/blob/v1.1.0/scipy). During updating, K u and f L were constrained to 0 for RA1 and PC units. w is constrained to 0 for SA1. The learning stepsize (α) was set to 0.55. The partial derivative of J (θ ) to each entry of θ was solved using centraldifference formula. All the parameters were initialized to zeros. To evaluate the parameter variation as shown in Table 1 , the whole training was repeated for 10 times. As seen in Figure 2A , after training of about 1000 iterations, the model for each type of tactile unit can be trained to be convergent (i.e., J (θ) remains stable). The fitting precision in Figure 2B represents root mean squared error between the predicted firing rates and observed ones. The fitting precision was calculated by taking mean square root of J (θ) /m across from the 1000th to the 1100th iteration, then multiplying with K f . The K f was set to 180, 200 and 300 for SA1, RA1, and PC units, respectively, referring the maximum firing rate of each afferent type (Muniak et al., 2007) . The fitting precision changing with the highest order of BPF is shown in Figure 2B . The fitting precision decreased significantly from order 1 to 3 for the PC units (P = 0.028, ANOVA), and from order 1 to 2 for the RA1 units (P = 0.022). While there was no significant decrease with the rise of order for the SA1 units (P = 0.486). For a trade-off between precision and fast computation, the highest orders for SA1, RA1 and PC receptor was set to 1, 2, and 3, respectively. The summary of parameters obtained by this training method at the preferred set of highest order of BPF are shown in Table 1 .
EXAMPLES OF IMPLEMENTING THE MODEL TO SIMULATE TACTILE AFFERENT RESPONSES OF SINGLE UNITS In the following section, we systematically compare simulated responses of tactile units with published data across wellestablished response properties in previous literature. We simulated the responses of single tactile unit to stimulus scaled as indentation depth. Simulations were performed on a personal computer with Intel processor i7-7700HQ, 8 GB of memory. The source code of current model and all simulations was written in Python3.6 using Numpy libraries and has been uploaded on a Github repository (https://github.com/ouyangqq/model_ of_single_tactile_unit). In all simulations, we used parameters given in Table 1 .
Reproducing Action Potentials Above Threshold In order to describe how the TCF and spike synthesizer covert mechanical indentation (x in ) into the action potential (v a ), the details of intermediate signals in response to typical sinusoidal stimuli are presented. As shown in Figure 3 , each type of tactile unit will not evoke spike activity until the amplitude of stimulus exceed its threshold. The evoked spike captures the typical shape of action potential as shown in Figure 1C .
Reproducing Selective Responses of Tactile Afferents The adaptation and frequency sensitivity are two basic selective response properties of a tactile unit. Hence a simulated experiment was conducted to evaluate the effectiveness of current model in predicting the selective responses recorded in previous neurophysiological experiments. A animation of simulating the selective responses of single tactile units can be found in Supplementary Material.
Tactile Sensory Adaptation The wave of mechanical indentation for SA1, RA1, and PC units were set referring to the literature of Mountcastle et al. (1966) , Talbot et al. (1968) and Knibestöl (1973) , respectively. As seen in Figure 4 , SA1 units respond to the onset and hold phase but typically not its offset stage. The firing rates in SA1 afferents increase almost linearly as indentation depth increases. The firing rate is higher during the dynamic phase of indentation than during steady indentation. When the probe is removed from the skin, the spike activity ceases. RA1 units respond strongly to onset and offset but typically adapt so quickly that they do not respond during static indentation. RA1 afferents can signal the rate at which the stimulus is applied and removed: rapid changes evoke a brief burst of high-frequency spikes, whereas slow changes evoke a longer-lasting, low-frequency spike train. PC receptor is highly sensitive to acceleration and higher derivatives. The PC afferents respond not only when the indentation is increasing, but also when the stimulus is retracted, however they are not very sensitive to punctate stimuli and completely independent of indentation velocity and amplitude. The above simulated responses of the three afferent types match well with the neurophysiological observed counterparts.
Frequency Characteristics of Neural Threshold The frequency sensitivity for each type of tactile unit is reflected in its frequency characteristics of neural threshold as illustrated in
Reproducing Spiking Timing As indicated in neurophysiological experiments investigating tactile encoding in the nerve, the cutaneous tactile afferents exhibit very precise and repeatable timed spike responses to vibratory stimuli (Talbot et al., 1968) . The importance of spike timing in tactile coding has since been established across a variety of tactile sensory modalities (Saal et al., 2016) , including vibratory frequency (Harvey et al., 2013) , surface texture (Weber et al., 2013) , surface curvature (Mackevicius et al., 2012) , and Observations [adapted from SA1, RA1 (Verrillo and Bolanowski Jr, 1986) , PC (Mountcastle et al., 1972) , HM (Gescheider et al., 1985) ]. (B) Prediction of our model. The human threshold (HM) is the minimum of the thresholds of the 3 mechanoreceptive types. The R2 in the legend is the coefficient of determination between the observed data in panel (A) and the predicted data in panel (B) for each afferent and for the human threshold (gray). direction of tangentially applied forces (Johansson and Birznieks, 2004) . To evaluate the performance of this model in reproducing spike timing, we carried out another simulation by using the recorded data of spiking responses to sinusoidal vibration from Muniak et al. (2007) . As shown in Figure 6A , the simulated responses to sinusoidal stimuli match well with their recorded counterparts. To quantify the timing precision of current model in predicting the recorded spiking responses, we computed the similarity between the simulated and recorded spike trains at different time scales using the metric of ISI (interspike interval) distance (Kreuz et al., 2007) . We could then determine how much we needed to jitter the recorded spike trains to achieve the level of temporal precision of the simulated responses using distance difference (dist. diff, referring to Saal et al., 2017) . The jittered spike trains was generated by sampling randomly from a zero-mean Gaussian distribution with a given SD and then adding to each recorded spike (Bensmaia et al., 2008) . We tested SDs ranging from 1 to 10 ms. As illustrated in Figure 6B , the model is worse if the averaged difference is greater than zero (horizontal black dotted line). The jitter value that averaged difference curve cross zero was defined as the precision of the model (vertical black dotted line Figure 6B ). We found that the current models of all afferent types achieve a temporal precision better than 6 ms. The PC models are the most precise, down to precision of about 2.5 ms, The SA1 and RA1 models achieve precisions ranging from 3 to 6 ms. In order to compare current model with the work (Saal et al., 2017) in terms of timing precision, we also reproducing the spiking trains in response to the same stimulus using code in work of (Saal et al., 2017) (http://bensmaialab.org/code/ touchsim/). The code of reproducing the spike trains as shown in Figure 6B are also available in our Github repository. As shown in Figure 6B , the current model performs relatively better the work of (Saal et al., 2017) , especially for SA1 afferents.
PERFORMANCE EVALUATION AND COMPARISON WITH PREVIOUS WORK Computational Efficiency To evaluate the computational efficiency of simulating massive numbers of tactile units, we present the averaged consuming time and the maximum number of tactile units that allow realtime simulation (MNTARS) at different sampling rates for each afferent type (Figure 7 ). When simulating multiple tactile units, the input, all intermediate or output variables were written in a two-dimensional Numpy arrays (see Figure 1D ), and the consuming time was measured with time stamp of Python. As seen in Figure 7B , the model runs in real time with about 300 units at a sampling rate of 4 kHz, and 2,000 units at rate of 1 kHz, and 4,000 units at rate of 500 Hz (running under the condition of 62% remaining free physical memory).
Comparisons With Previous Work In order to compare with the previous spike neuron models, we also implemented the Hodgkin-Huxley, FitzHugh-Nagumo, Izhikevich Model, and IF models referred from the literature (Hodgkin and Huxley, 1952; Fitzhugh, 1961; Izhikevich, 2003; Brette and Gerstner, 2005) , respectively. The codes of these compared models were rewritten with Python and available in our Github repository. The NSM model was not implemented for comparison, since its code is not available. Comparing firing spike reproduction by the previous spiking neuron models, the spike waveform generated by spike synthesizer matches the typical recorded ones almost perfectly (see Figure 8A ). Although the spike synthesizer did not show computational efficiency over some spiking neuron models (e.g., FN, IF, IZ, see Figure 8B ) especially at high sampling rate, improvement of matching recorded spiking shape (6th trace in Figure 8A ) over these models is dramatic. In addition, the spike synthesizer can be implemented to accurately reproduce action potential of any shape by changing base wave in Figure 1B . A multifaceted comparisons between this model and previous related work are shown in Table 2 . Compared to the NSM model and the Work of (Saal et al., 2017) , our model have less parameters, but it can be implemented to characterize 3 major types of tactile units, and allow for a faster computation of reproducing most of their response properties with higher timing precision.
DISCUSSIONS In the present work, we simulated the responses of single tactile receptors and their afferents activated by a punctate or vibrating stimuli. The current model creates a biologically plausible response using many characteristics taken from the known neurophysiological literature. As depicted in Figure 5B , the adaptation properties of simulated units (Figure 4 ) create accurate frequency characteristics of neural threshold (Figure 5B ). It supports the hypothesis that frequency characteristics of neural threshold is mostly due to the adaptation properties (Bolanowski et al., 1988) . As illustrated in Figure 6 , we found a high temporal agreement between the observed spike timing and that predicted by this model, which can be attributed to the training method using neurophysiological dataset. Compared to model developed by Saal et al. (2017) , this model make use of frequency modulator as illustrated in Figure 1B to generate action potentials whose firing rate is linear to the output of TCF model. Therefore, the current model can be simplified easily and trained accurately, which allows for quickly simulating thousands of tactile units with high timing precision (see Figures 6, 7B ). The model developed by Saal et al. can also be implemented to reproduce spiking responses with millisecond precision for the 3 types of tactile units, and runs in real time with 300 units at a rate of 300 Hz (Saal et al., 2017) . Their work includes an IF model, and the continuum mechanics (CM) model to simulate the skin mechanics. As illustrated in Figure 6 , the current model achieve almost same timing precision for PC units, but performs better for SA1 and RA1 units. As seen in Figure 7B and Figure 8B , the current model allow for the realtime simulation of about 4,000 tactile units at a rate of 500 Hz and reproducing the accurate spike shape of tactile afferents. The spiking synthesizer can be implemented to reproduce biological action potentials of different tactile neuron types by replacing different base waves (see Figure 1C ). Since there is a diversity in shape of action potentials in peripheral and central neurons, the spiking synthesizer may be extended simulate other sensory neurons (Bean, 2007) . Compared to model developed by Saal et al. the current work lack of CM model to simulate skin mechanics. The CM model adopted by Saal et al is fast to compute (Sripati et al., 2006) , thus the current model combining with the CM model will perform better in biological compatibility and computational efficiency than the model by Saal et al. It should be noted that the simulations were mostly compared to responses recorded from nerves innervating the monkey fingertip, but the model can also be expanded for the human by only adjusting the mechanotransduction coefficient (A s ), since the individual tactile afferents in human and macaque behave nearly identically in basic response properties (Muniak et al., 2007) . Interestingly, we also found that all three types of mechanoreceptors were sensitive to a stimulus with a sudden ramp change. It is common among sensory systems for dynamic stimuli to generate short, but strong, responses. This can be seen in vision (Bisley et al., 2004) , and the auditory system (Flint et al., 2011) , as well as in the tactile system. This phenomenon could be associated with orienting reflex, which is an organism's immediate response to a change in its environment. Animals constantly focus attention to changes of the stimulus, which is an important way to survive.
APPLICATIONS A mathematical model to quantitatively characterize tactile unit in response to stimuli and to output a response in a biological code has great potential. For instance, the proposed model can be a powerful tool to investigate the sense of touch. Indeed, recording the responses of human or monkey afferents is technically challenging and only yields responses at a time. The model could also be used to create hypotheses about how more complex stimuli are encoded in the tactile periphery, which can then be tested in the human or animal model. The whole hand contains about 17,000 tactile units (Johansson and Vallbo, 1979) , which work together to transmit information about shape, texture and movement. After a certain simplification, our model allows for real-time simulating the responses of tactile afferents in whole hand. The model can be used as a tool to investigate shape and texture encoding in nerve system. The PC afferents play a role in the perception of fine textures whose elements are too small and closely spaced to be processed spatially (Hollins et al., 2002) , and even for the perception of relatively coarse textures (Cascio and Sathian, 2001; Gamzu and Ahissar, 2001) . This model lays the groundwork for researchers to investigate the interactive effect between PC and SA1 afferent responses to fine textures, which provide a valuable insights to improving realism of rendering virtual fine texture with haptic devices. An exciting use of this model is in brain-machine interfaces. Using a model such as this, signals transduced by sensors located on a prosthesis could be converted into patterns of neural activity, which could then be sent to a peripheral nerve to accurately represent how the nerve in hand would respond to skin indentation. In addition, the spiking synthesizer could be used to convert any spike rate input into a burst of biological action potentials with high timing precision, which may improve the biocompatibility of the current sensory prosthesis. The current model may also leads to new ways of designing highly realistic tactile interfaces such as neurorobots and bionic hands (Bologna et al., 2001) .
LIMITATIONS OF MODEL This model faithfully reproduces key response properties of tactile afferents, but it is also subject to some limitations. First, while we were able to simulate single-afferent response for SA1, RA, and PC afferents, we did not simulate SA2 fibers, since there are very few neurophysiological experimental data for fitting the SA2 model, and the primary function of SA2 fibers is thought to be encoding stretch, rather than indentation (Johnson, 2001) . Second, this model treats the tactile unit as a singleunit isolated from other units, when in reality, it is not. Histological studies reveal that the connections are ubiquitous in tactile afferents, with single afferent fibers innervating multiple receptors and single receptors receiving multiple afferent fibers (Par et al., 2002) . However, the model can be extended to accommodate afferent branching, especially when mechanisms by which input from the multiple receptors is integrated to evoke the afferent responses are better understood by us Lesniak et al. (2014) . Third, the current model rapidly recreates the temporal firing characteristics of tactile units with high temporal precision, however the mimicry of their spatial characteristics is not addressed. The spatial characteristics of spikes is reflected in afferent population response (Yoshioka et al., 2001; Weber et al., 2013) . To simulate population response, parameters such as the skin elasticity and relative positions between the contact points and the receptors on the contact area are required. The skin biomechanics which includes these parameters can be included in has been proven to be important to simulating tactile afferent response (Birznieks et al., 2010) . Although skin mechanics is not considered in this study, the current model is also useful. As shown in Figure 1D , all the signal variables were defined as two dimensional arrays to set an interface for simulating population responses. By adding an accurate skin mechanics model to compute the skin indentations from stimuli, the current model has the capability of reproducing afferent population response to stimuli of different shape and size with high timing precision.
CONCLUSION The current work provides a pieces of Python code to accurately reproduce the selective responses of single tactile units in a biological pattern using an electromechanical circuit model. This model has the potential of giving us a better understanding of afferent firing patterns in response to skin indentations. This work may provide valuable guidance to designing prostheses with tactile feedback, enhancing the realism of haptic rendering of virtual tactile stimuli, and building a digitized human hand with physiological response in a virtual surgical system. Although the current work has mimicked the responses of single tactile receptors and afferents comprehensively, it is not enough to recreate the tactile afferent population responses. Complex tactile stimuli such as texture, shape, roughness of a grating, and edge orientation, are encoded in population responses (Khalsa et al., 1998; Hollins and Bensmaïa, 2007; Weber et al., 2013; Suresh et al., 2016) . In the future, we will address these issues by constructing a computing model of population responses. This is beyond the scope of the current work but we think this might allow for a better understanding of peripheral representation of tactile stimuli. FIGURE 1 | 1 FIGURE 1 | Schematic diagram of current model. (A) Overall model of single tactile unit. x stand for the indentation displacement produced in the skin. (B) Schematic diagram of spike synthesizer. (C) Typical shape of a spike. (D) Source code of implementing schematic diagram. The 'f_sp()' is the function of the waveform shown in (C).
FIGURE 2 | 2 FIGURE 2 | (A) Averaged loss value (J (θ) ) changing with iterations under different highest order of BPF (n). (B) Fitting precision changes with highest order of BPF. Error bars represent the standard deviation. The difference between the fitting precisions at two orders was evaluated using post-hoc tests.
FIGURE 4 | 4 FIGURE 4 | Adaptation properties of different types of tactile units. The recorded data of SA1, RA1, and PC are adapted from Mountcastle et al. (1966), Talbot et al. (1968), and Knibestöl (1973), respectively. (A,B) Recorded (left) and simulated (right) responses of single SA1 unit to ramp-and-hold indentations at different indentation depths, and single RA1 unit to ramps approaching indention of 850 um at different speeds. The stimulus amplitude and time course are shown in the lower trace of each pair; the upper trace shows the action potentials recorded from the sensory nerve fiber in response to the stimulus. (C) Recorded (left) and simulated (right) responses of a PC unit to stimulus with reduction of indentation amplitude. The upper traces show the recorded action potential for five amplitude levels in descending order downwards, and the lower traces show the analog signals of the corresponding stimuli.
Figure 5A . 5A In current model, since the firing rate of the outputting spikes is determined by the output signal of v nf , the predicted frequency characteristics of neural threshold for each afferent type can been a calculated according to equation (3). As shown in Figure5B, the human thresholds for vibration closely match those of the most sensitive afferent fibers in each range (R 2 = 0.94).
FIGURE 5 | 5 FIGURE 5 | The average frequency-threshold curves of single-unit. (A) Observations [adapted from SA1, RA1 (Verrillo and Bolanowski Jr, 1986) , PC (Mountcastle et al., 1972) , HM (Gescheider et al., 1985) ]. (B) Prediction of our model. The human threshold (HM) is the minimum of the thresholds of the 3 mechanoreceptive types. The R2 in the legend is the coefficient of determination between the observed data in panel (A) and the predicted data in panel (B) for each afferent and for the human threshold (gray).
FIGURE 6 | 6 FIGURE 6 | Evaluation of spike-timing precision for each afferent type. (A) Recorded (gray dotted marks) and simulated spike trains of 5 sampled tactile units for each afferent type in response to a sinusoidal stimulus of 20 Hz vibration (black solid line) with amplitudes of 35 (left), 130 (center) and 250 um (right). Recorded spike trains and the stimulus were adapted from Muniak et al. (2007). (B) Difference in the spike distance between the simulated and jittered spike trains to recorded spike trains as a function of jitter SD. The vertical black dotted line localizing at the cross of zero horizontal line and the quadratic fitting curve of current model determine the timing precision.
FIGURE 7 | 7 FIGURE 7 | Evaluation of computation efficiency for simulating population units. (A) Averaged consuming time for simulating each type of tactile unit for 1 second at different setting number under different sampling rate. Error bar represent the standard deviation of the mean. (B) MNTARSs at different sampling rates. The MNTARS was evaluated as the setting number at cross of consuming time curve and horizontal dotted line in panel (A).
FIGURE 8 | 8 FIGURE 8 | Performance comparison between different spiking neuron models. (A) Reproduction of spiking waveform (200 ms frame) using our spike synthesizer and previous neuron models (from top 1 to 5th trace), their applied current (i a , nA) is shown in gray curve in each simulated trace. The recorded typical spiking waveform reprinted from Hodgkin et al. (1952), are depicted in bottom. (B) Consuming time of converting applied current into action potential for simulation in left figure at different sampling rate using different neuron models. Error bars represent the standard deviation.
TABLE 1 | 1 Summary of parameters (mean ± SD). Parameters Mean in this model SA1 RA1 PC K u Weight of LPF 0.094 ± 0.031 0 0 K b1 Weight of 1st order BPF 0.205 ± 0.008 0.232 ± 0.021 0 K b2 Weight of 2rd order BPF 0 0.0031 ± 0.00021 0.128 ± 0.014 K b3 Weight of 3th order BPF 0 0 0.00111 ± 0.00011 f BL Lower cutoff Frequency of BPF (Hz) 8.01 ± 0.21 60.10 ± 1.61 80.40 ± 1.82 f BH Upper cutoff Frequency of BPF (Hz) 10.03 ± 0.41 80.09 ± 2.32 220.02 ± 4.21 f L Upper cutoff Frequency of LPF (Hz) 100.20 ± 0.72 0 0 A s Coefficient of transducer (V/mm) 3.80 ± 0.13 44.00 ± 1.33 0.36 ± 0.03 w Negative contribution for rectifying 0 0.015 ± 0.002 0.212 ± 0.021 K f Firing-rate encoding coefficient 180 200 300
TABLE 2 | 2 A comparison between the contributions of this work and the related works. Models Parameter MNTARS TP Properties allowed to be simulated Type of afferent fiber number (units) (ms) FC AT AP SA1 RA1 PC This work 10 ≈4000 <6 Y Y Y Y Y Y Work of Saal et al., 2017 13 <350 <7 Y Y Y Y Y NSM model Biswas et al., 2015 >20 Y Y Y N N Y TP, Timing precision; FC, Frequency sensitivity; AT, Absolute threshold; AP, adaptation properties; Y, supported; N, unsupported.
Frontiers in Neuroinformatics | www.frontiersin.org
April 2019 | Volume 13 | Article 27
|
10.1109/tnnls.2023.3236635
|
cc-by
| null | null |
openalex
|
Neuropsychological studies suggest that cooperative activities among different brain functional areas drive high-level cognitive processes. To learn the brain activities within and among different functional areas of the brain, we propose local-global-graph network (LGGNet), a novel neurologically inspired graph neural network (GNN), to learn local-globalgraph (LGG) representations of electroencephalography (EEG) for brain-computer interface (BCI). The input layer of LGGNet comprises a series of temporal convolutions with multiscale 1-D convolutional kernels and kernel-level attentive fusion. It captures temporal dynamics of EEG which then serves as input to the proposed local-and global-graph-filtering layers. Using a defined neurophysiologically meaningful set of local and global graphs, LGGNet models the complex relations within and among functional areas of the brain. Under the robust nested crossvalidation settings, the proposed method is evaluated on three publicly available datasets for four types of cognitive classification tasks, namely the attention, fatigue, emotion, and preference classification tasks. LGGNet is compared with state-of-the-art (SOTA) methods, such as DeepConvNet, EEGNet, R2G-STNN, TSception, regularized graph neural network (RGNN), attentionbased multiscale convolutional neural network-dynamical graph convolutional network (AMCNN-DGCN), hierarchical recurrent neural network (HRNN), and GraphNet. The results show that LGGNet outperforms these methods, and the improvements are statistically significant ( p < 0.05) in most cases. The results show that bringing neuroscience prior knowledge into neural network design yields an improvement of classification performance. The source code can be found at https://github.com/yi-ding-cs/LGG.Length of the hidden output of GCN layers.
Υ() Flatten operation.
I. INTRODUCTION B RAIN-COMPUTER interface (BCI) enables the brain to communicate with machines directly using electroencephalography (EEG) . A typical BCI system consists of a data acquisition module, a preprocessing module, a classification module, and a feedback module . BCI has a wide range of applications in the real world, such as robot controlling , stroke rehabilitation , and emotion regulation for mental disorders , . Compared with traditional machine learning methods , , , , deep learning methods achieved superior performances in different tasks of BCI, such as classification of motor imagery , , , , mental attention classification , , , emotion recognition , , , , and mental workload detection . However, most of the previous studies highly rely on manually extracted EEG features, such as power spectral density (PSD) , and differential entropy (DE) , , . With the feature-extracting ability of convolutional neural networks (CNNs), directly learning from EEG becomes reliable , , . There are mainly two types of information to This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ be learned in EEG, temporal and spatial information. The temporal information is well studied by the 1-D CNNs , and multiscale 1-D CNNs . For spatial information, previous methods either learn global spatial information using 1-D CNNs along electrode dimension , , or apply small 2-D CNN kernels on image-liked EEG 2-D maps , , to extract local spatial information separately, which may not learn the spatial information effectively. EEG signals can be naturally regarded as graph-structured data, with each electrode being the node and spatial relations or correlations among electrodes being the edges. A graph neural network (GNN) with proper adjacency relations can jointly learn the localized and global spatial patterns in EEG. Incorporating the prior knowledge from neuropsychological studies into GNN design has huge potentials in mental states decoding from EEG. The brain is a complex network with a hierarchical spatial and functional organization at the level of neurons, local circuits, and functional areas . Different functional areas correlate to certain brain functions while not working independently . Activating one particular brain region also tends to activate other regions in the group . How to design neurophysiologically meaningful networks to effectively model the brain activities within and among different functional areas of the brain becomes crucial. Some studies , used a global adjacency matrix with learnable connections which paid less attention to the localized activities in each functional area. Regularized graph neural network (RGNN) built the connections according to the spatial distance among electrodes. Although it added fixed global connections to improve the decoding performance, the complex relations among functional areas were not learned capably. To address the above problems, we propose to define the EEG data as a local-global graph (LGG) whose local graphs belong to the different functional areas of the brain according to neurological knowledge , . The nodes in each local graph are fully connected because they reflect the brain activities within each brain functional area. The edges of local graphs, or the global connections among local graphs, reflect the complex functional connections among different brain functional regions. To extract more information-rich representations from EEG as the node attributes in the proposed LGG representations of EEG, a temporal convolutional layer with multiscale 1-D convolutional kernels is adopted . A kernel-level attentive fusion layer is further designed to fuse the learned temporal representations with attention. For graph connection learning, a local-graph-filtering layer and a global-graph-filtering layer are proposed to learn the brain activities within and among different local graphs. In the localgraph-filtering layer, the attributes of the nodes are attentively aggregated into one hidden embedding which represents the activity of the local graph. For the global-graph-filtering layer, an instance-specific similarity matrix is proposed as the base adjacency matrix of the global graph. Inspired by dynamical graph CNN (DGCNN) , a learnable adjacency mask is further utilized to select the global connections attentively via backpropagation during the training process. We propose general, frontal, and hemisphere LGG definitions of EEG based on neurophysiological evidence of associations among brain areas for different mental tasks. The general LGG is defined according to the 10-20 system that groups the electrode based on the location of electrodes on functional areas . In the frontal LGG, the frontal region is further divided into smaller local regions which are symmetrically located on the left and right hemispheres to learn asymmetric patterns in emotion . In the hemisphere LGG, the symmetrically located subgraphs exist in all the functional areas. In this article, we propose LGG network (LGGNet) that integrates all the aforementioned learning blocks to model the activities within and among brain functional areas for mental state classification. LGGNet was evaluated on four classification tasks, attention, fatigue, emotion, and preference classification, using three publicly available benchmark datasets, the attention dataset , the fatigue dataset , and the dataset for emotion analysis using EEG, physiological and video signal (DEAP) dataset , respectively. The proposed LGGNet was compared with several state-of-the-art (SOTA) methods in the BCI domain. From the experiment results, LGGNet achieved the highest accuracies and F1 scores among the compared SOTA methods in most of the classification experiments. Furthermore, ablation studies were conducted to understand the importance of kernel-level attentive fusion, local-and global-graph-filtering layers in LGGNet. To evaluate the effectiveness of involving neuroscientific prior knowledge in LGGNet, the effect of building EEG as LGGs as well as the differences among different graph definitions were analyzed. After that, extensive visualization experiments were conducted to better understand what the network learned from EEG. The most informative region of the data identified by the network was visualized using saliency maps . The learned adjacency matrices for different cognitive tasks were visualized as well. The major contributions of this work can be summarized as follows. 1) Proposed LGGNet, a neurologically inspired GNN, to learn the brain activities within and among different brain functional areas. 2) Three different types of LGGs, namely the general, frontal, and hemisphere LGGs, were proposed to study the effects of different graph definitions on different cognitive tasks. 3) The proposed method was compared with DeepCon-vNet (2017) , EEGNet (2018) , R2G-STNN (2019) , TSception (2020) , RGNN (2020) , attention-based multiscale CNN-DGCNN (AMCNN-DGCN) (2021) , hierarchical recurrent neural network (HRNN) (2021) , and GraphNet (2021) on three publicly available datasets for four different types of cognitive tasks: attention, fatigue, emotion, and preference classification. 4) Extensive ablation studies and analysis experiments were conducted to better understand LGGNet. The remainder of this article is organized as follows. Some related work is given in Section II. In Section III, the proposed LGGNet is introduced. In Section IV, the dataset and experiment settings are presented. The result and discussion are provided in Section V. Finally, we conclude the article in Section VI.
II. RELATED WORK
A. Different Representations of EEG Data EEG data have two dimensions: channels (EEG electrodes) and time. The channel dimension reflects the brain activities across different functional areas due to different locations of electrodes on the surface of the human's head. The channel refers to the EEG electrodes if not specified. The time dimension contains the changes in brain activities over time. There are three types of EEG representations commonly used in recent studies, namely 2-D time-series, images, and graphs. For 2-D time-series formats, the network input layer typically consists of temporal convolutional layers to extract temporal information channel by channel and spatial convolutional layers to extract spatial information , , . Another type of EEG representation is the image. In this, the electrodes are rearranged into a 2-D frame based on their relative locations on the brain surface, and the raw data or features of each electrode will be the third dimension of the 2-D map , . Recently, many studies , , have represented EEG data as graphs. In these studies, EEG signals are treated as graphs, with the electrodes being the node and spatial distance or correlations being the edges.
B. Graph Neural Networks A graph is represented as G = (V, E), where V is the set of nodes, and E is the set of edges. v i ∈ V denotes a node, and e i, j = (v i , v j ) ∈ E denotes an edge. The adjacency matrix A is derived as an n × n matrix with A i, j = 1 if e i, j ∈ E and A i, j = 0 if e i, j / ∈ E. A graph, also known as attributed graph, may have node attributes X, where X ∈ R n×d is a node feature matrix with x v ∈ R d representing the feature vector of a node v. A graph can be a directed graph or an undirected one. The adjacency matrix of a directed graph may not be asymmetric if a single-direction connection exists (e.g., e i, j = e j,i ). The adjacency matrix of an undirected graph is symmetric, and A = A T . GNN was proposed to deal with the graph-structured data. Graph CNNs (GCNNs) extended the convolution operation to graph in the spectral domain. It can generate a node representation by aggregating its features and neighbors' features. Kipf and Welling proposed a scalable GCNN, which can encode both localgraph structure and the feature of the node with improved computational efficiency.
C. Graph Neural Networks for EEG We review some related works that use GNNs to decode EEG signals. Jang et al. defined the connections as both spatial locations and correlations among electrodes to do video classification via EEG graphs in 2018. In the same year, Song et al. designed DGCNN for EEG emotion recognition with a trainable adjacency matrix. Lian et al. refined the graph topology by incorporating the dynamically learned connection weights based on attention and gating mechanisms. GCB-Net also utilized a trainable adjacency matrix, and the broad learning system was further applied to learn shallow and deep features. Zhong et al. defined the adjacency matrix according to the spatial distance and added some global connections according to asymmetry in neuronal activities. GraphNet utilized GCN with a distance-based adjacency matrix to decode mental attention states. Instead of learning from hand-crafted features, AMCNN-DGCN learned from EEG directly using multiscale CNN kernels. After that, GCN layers with a trainable adjacency matrix were applied to learn the spatial relations among electrodes. Although many GNNs were proposed for EEG decoding, most of them did not model the brain activities within and among different functional areas.
III. LGGNET FOR BCI In this section, LGGNet is introduced. Nomenclature illustrates the notations used in this section. As shown in Fig. 1, LGGNet has two main functional blocks, a temporal learning block and a graph learning block. The temporal convolutional layer in the temporal learning block aims to learn dynamic temporal/frequency representations from EEG directly instead of manually extracted features with the help of a kernellevel attentive fusion layer. The graph learning block contains two layers, namely the local-and global-graph-filtering layers. The local-graph-filtering layer learns the brain activities within each neurophysiologically meaningful local region, after which the global-graph-filtering layer with a similaritybased trainable adjacency matrix will be applied to learn complex relations among different local regions.
A. Temporal Learning Block Temporal learning block has two modules: temporal convolutional layer and kernel-level attentive fusion layer. 1) Temporal Convolutional Layer: The multiscale temporal convolutional layer utilizes parallel multiscale 1-D temporal kernels (T kernels). In order to learn dynamic-frequency representations, the length of the temporal kernels is set in different ratios of the sampling rate f S . The ratio coefficient is denoted as α k ∈ R, where k is the level of the temporal convolutional layer. k will vary from 1 to K (α = 0.5, K = 3, in our study). Hence, the size of T kernels in kth level, denoted by S k T , can be defined as S k T = 1, α k f S , k ∈ [1, 2, 3]. ( 1 ) Given the preprocessed EEG data X i ∈ R c×l , i ∈ [1, . . . , n], where n equals the number of EEG samples, c is the EEG channel number, and l is the sample length in the time dimension, three multiscale temporal kernels are applied parallelly to learn dynamic temporal/frequency representations. Instead of using ReLU () as TSception , we use the logarithmic of the average pooled square of the representations as to learn the power features, which are well-studied EEG features in the BCI domain. The 1-D CNN layer serves as digital filters which can output filtered signals in different frequency bands . By squaring the filtered signals, we can get the power of them. An average pooling layer acts as a window function to calculate the averaged power in shorter segments. Then, a logarithmic activation is applied as which shows adding the logarithmic activation can help to improve the performance. Let Z k temporal ∈ R t×c× f k denote the output of the kth level temporal kernel, where t is the number of T kernels, and f k is the feature length. Z k temporal is defined as Z k temporal = log F AP square F Conv1-D X i , S k T ( 2 ) where F Conv1-D (X i , S k T ) is the convolution operation using T kernel of size S k T on X i , square () is the square function, F AP () is the average pooling operation, and log () is the logarithmic function. The pooling size and step of the F AP () in this power layer were (1, 128) and (1, 0.25 * 128 = 32) for the attention and the fatigue dataset, the pooling size for DEAP was set as (1, 16) since DEAP had more data that needed a deeper model to learn. The output of all levels' T kernels will be concatenated along the feature dimension. Hence, the output of the multiscale temporal convolutional layer for X i , Z i MS ∈ R t×c× f k , can be calculated by Z i MS = Z 1 temporal , . . . , Z K temporal (3) where () is the concatenation operation along the feature (f) dimension. 2) Kernel-Level Attentive Fusion: After concatenation of the output from different level T kernels, a one-by-one convolutional layer is adopted as a kernel-level attentive fusion layer to fuse the features learned by different kernels. Batch normalization is utilized before and after the one-byone convolution to reduce the internal covariate shift effects. The number of one-by-one kernels is set as t. Leaky rectified linear unit (ReLU) is utilized as the activation function. After that, an average pooling layer is utilized to downsample the learned representations. After batch normalization, the fused representations from different one-by-one kernels are then flattened for each EEG channel as its node attribute in EEGgraph representation that will be introduced in Section III-B1. This reshaping process is shown in Fig. 1(a) . Hence, the attentively fused temporal representation of each X i , Zfuse , is calculated by Zi fuse = F bn F AP L-ReLU F fuse F bn Z i MS ( 4 ) where F bn () is the batch normalization function, F fuse () is the one-by-one convolution function, and the L-ReLU () is the leakey ReLU() activation function. The kernel and step sizes of F AP () are both (1, 2). The Zi fuse ∈ R c×t×0.5 * f k is reshaped to Z i fuse ∈ R c×t * 0.5 * f k to build the attribute of each node (EEG channel) in the EEG-graph representations Z i fuse = F reshape Zi fuse . ( 5 )
B. Graph Learning Block 1) Defining LGGs of EEG: In this section, three types of LGG representations are constructed based on neuroscience findings , , , namely general LGG G g , frontal LGG G f , and hemisphere LGG G h . Given Z fuse ∈ R c×t * 0.5 * f k , each electrode is regarded as one node in the EEG graph, and the learned dynamic temporal representations of electrodes are This article has been accepted for inclusion in a future issue of this journal. Content is final as presented, with the exception of pagination. regarded as the node attributes. To learn more information on graph data, the adjacent relations among nodes are very important. To effectively define adjacent relations, several neuroscience findings are taken into consideration. First, we define a general LGG. Human brains have several functional regions which will be active during different cognitive processes . EEG electrodes are placed on scalp surfaces according to the 10-20 system that groups channels according to the location on different functional areas of the brain. We define general LGG, G g , based on the different functional areas of the brain according to the 10-20 system. It is shown in Fig. 2
(a). LGGNet using the general LGG definition is regarded as LGGNet-G and may be used for more generalized BCI classification tasks. The frontal LGG is further defined based on several neuroscience findings on cognition and emotion studies. The frontal lobe is responsible for high-level behaviors, such as thinking, attention, and emotions , . The frontal asymmetries of EEG appear both on emotional processes and attentional bias to threat . Hence, the frontal area is further split into several smaller local graphs which are symmetrically located on the left and right frontal hemispheres to learn more discriminative information. The frontal LGG, G f , is shown in Fig. 2(b) . For the hemisphere LGG, we adopt the definition in , which has symmetrical subgraphs on the left and right hemispheres for all the functional areas. The hemisphere LGG, G h , is shown in Fig. 2(c) . We reorder the EEG channels according to the above LGGs. The channels within each local graph are next to each other so that the aggregation operation can be applied in the localgraph-filtering layer Z i reorder = F reorder Z i fuse . ( 6 ) 2) Local-Graph-Filtering Layer: In order to learn the local brain activities, a local-graph-filtering layer is proposed to attentively aggregate the learned representations within each local graph, Z reorder ∈ R c×t * 0.5 * f k . In this section, the local connections are defined. Then the local-graph-filtering layer is introduced. The electrodes within one local graph are fully connected. The brain consists of local circuits and functional areas . indicates that the strength of connections among brain regions decays as the physical distance increases. Hence, we hypothesize that different electrodes within a subgroup can reflect the similar brain activities of the corresponding functional areas. The local adjacency matrix A local is defined as
Salvador et al.'s study A local = ⎡ ⎢ ⎣ 1 1 . . . . . . . . . 1 1 ⎤ ⎥ ⎦ (7) where all elements are 1. The size of the A local depends on how many channels are within the local graph. There are two steps in the local-graph-filtering layer: Localgraph filtering and local representation aggregating. Given the trainable local-graph-filtering matrix W local ∈ R c×t * 0.5 * f k , and local-graph-filtering bias vector b local ∈ R c×1 , the localgraph-filtering weights will be assigned to the representation of each electrode by Z i filtered = ReLU W local Z i reorder -b local ( 8 ) where Z i local = F aggregate Z i filtered = F aggregate Z 1 , . . . , Z R T = ⎡ ⎣ 1 P 1 P 1 p=1 z p 1 , . . . , 1 P R P R p=1 z p R ⎤ ⎦ T = h 1 local , . . . , h R local T ( 9 ) where p is the index of nodes in each local graph, and h local is latent representations of local graphs. 3) Global-Graph-Filtering Layer: The graph convolution on the global graph is designed to learn the complex relations among local graphs. We first define the global connections after which the details about the global-graph-filtering layer are presented in this part. The global connection is defined based on the relations among local graphs. Neuroscience studies suggested that activating one particular brain region also tends to activate other regions in the group for the high-level cognitive process . The relations among local graphs are utilized as the edges in the global graph. The dot products between local graph representations for each EEG instance are calculated to reflect the relations among local graphs. Note that, the similarity adjacency matrix is dynamic and instance-specific. We assume the global connection is undirected because the relation between two local graphs is mutual. The basic adjacency matrix of the global graph, A global-base ∈ R R×R , is symmetric and can be defined as A global-base = ⎡ ⎢ ⎣ h 1 local h 1 local h 1 local h R local . . . . . . . . . h 1 local h R local h R local h R local ⎤ ⎥ ⎦ (10) where is the dot product. Due to the complex relations among brain functional areas, a trainable attentive mask is adapted to emphasize the most important connections in the instance-level similarity adjacency matrix. Note that the trainable mask is also symmetric because the global adjacency matrix is undirected. The trainable attentive mask, M ∈ R R×R , can be defined as M = ⎡ ⎢ ⎣ w 1,1 w 1,R . . . . . . . . . w R,1 w R,R ⎤ ⎥ ⎦ (11) where w are the trainable parameters, and w 1,R = w R,1 . Self-loops are added after applying the trainable mask to the basic global adjacency matrix. Because adding self-loops after applying the trainable mask can maintain the strength of the self-loops since the values in the trainable mask are generally small. The ReLU activation function is applied to make the adjacency matrix non-negative. Hence, the final global adjacency matrix can be calculated as A global = ReLU A global-base M + I ( 12 ) where I ∈ R R×R is the identity matrix. Given the global adjacency matrix, A global ∈ R R×R described in (12) , a GCN layer is adopted to learn the global-graph representations. The normalized adjacency matrix, A global , can be calculated by A global = D -1 2 A global D -1 2 (13)
Algorithm 1 LGGNet Return: pr ed Input: EEG data X i ∈ R c×l ; where D = q A p,q global is the degree matrix of the A global . Before the global-graph filtering, batch normalization, F bn (), is applied. In LGGNet, the number of global GCN layers is set to be one. Let the projecting weight matrix of GCN layer be W global ∈ R f ×h , where h is the length of the hidden output after GCN, and the trainable bias vector be b global ∈ R h×1 . The global-graph filtering of Z i local can be calculated by Z i global = ReLU A global F bn Z i local W global -b global . ( 14 ) After getting the globally filtered representation, batch normalization is applied. Then the flattened representation will be fed into a linear layer to generate the final classification output as Output = softmax WF dropout Υ F bn Z i global + b ( 15 ) where the Υ() is the flatten operation, W is the trainable weight matrix, and b ∈ R n classes ×1 is the bias term (n classes is the number of classes which is two in this article). The structure of graph learning block is summarized in Table I . It shows the operations, input, and output of each module. A single output sample whose size is (c × f ) from the temporal learning block is used for the illustration. Finally, the proposed LGGNet can be summarized in Algorithm 1.
IV. EXPERIMENTS
A. Datasets Three publicly available datasets of different tasks were utilized to evaluate the proposed LGGNet: the attention dataset for attention classification, the fatigue dataset for fatigue classification, and the DEAP dataset for emotion and preference classification, respectively.
TABLE I STRUCTURE OF LOCAL-AND GLOBAL-GRAPH FILTERING The attention dataset 1 is a multimodal brain-imaging dataset to measure three cognitive tasks of healthy subjects. The discrimination/selection response task (DSR) was involved in this article for attention classification. Twentysix subjects participated in the experiment. The session among the three was utilized for each subject to avoid the effects of cross-session variance. There were several series of attention task periods (40 s) and rest periods (20 s) in each session. Twenty-eight EEG channels and two electrooculography (EOG) channels were recorded with a sampling rate of 1K Hz. The fatigue dataset 2 provides the EEG signals to measure the cognitive fatigue states of the driver during a 90-minlong driving task in a virtual reality (VR) driving environment. Twenty-seven subjects participated in the data collection experiments. The subjects were introduced to keep the car cruising in the center of the lane while random lane-departure events were induced. Thirty-two channel EEG signals were collected with a sampling rate of 500 Hz. DEAP 3 is a multimodal human affective states dataset, including EEG, facial expressions, and galvanic skin response (GSR). Forty 1-min-long emotional music videos were used to induce different emotions to the subject. Before each trial, there was a 3-s baseline. Subjects provided their selfassessments on arousal, valence, dominance, and liking after each trial, using a continuous nine-point scale. The valence and liking dimensions were utilized for the emotion and preference classification tasks in this article. Thirty-two subjects participated in the data collection experiments. Thirty-two channel EEG signals were recorded with a sampling rate of 512 Hz.
B. Preprocessing EEG signals with several preprocessing operations were used as the input samples of the neural networks instead of hand-crafted features. For the attention dataset, a band-pass filter from 0.5 to 50 Hz was applied to remove low and high-frequency noise as . EOG was removed using the automatic independent component analysis (ICA) EOG removal method in the magnetoencephalography and electroencephalography (MNE) toolbox . Then the data were downsampled to 200 Hz. Following , only the first half of each attention trial was utilized to balance the samples between attention and 1 http://doc.ml.tu-berlin.de/simultaneous_EEG_NIRS/ 2 https://figshare.com/articles/dataset/MultichannelEEGrecordingsduringasus tained-attentiondrivingtask/6427334 3 http://www.eecs.qmul.ac.uk/mmv/datasets/deap/index.html inattention (rest). Each trial was further segmented into 4-s segments with a 50% overlap. For the fatigue dataset, the officially preprocessed EEG dataset was used in this article. The raw EEG signals were band-passed from 1 to 50 Hz. Eye blinks were removed by visual checking. The automatic artifact removal (AAR) method in EEGLab was used to remove ocular and muscular artifacts. The processed data were downsampled to 128 Hz as . For fatigue level calculation, we also followed . The 3s' EEG data before the onset of the lane-departure events were used as EEG trials. Reaction time (RT) was utilized to measure the fatigue level for the EEG trials. RT was defined as the time from the onset of the lane-departure event to the onset of the counter-steering event. The RT of one trial was defined as local RT, denoted by RT l . The global RT (RT g ) of the one trial was the mean of the local RTs of all the trials within a 90-s window before the current trial. The fifth percentile of all local RTs in the entire session was selected as an alert RT, RT a . Let 0 be the label of the fatigue class, and 1 be the nonfatigue class, the labeling process can be defined as y = 0, RT l > 2.5 * RT a && RT g > 2.5 * RT a 1, RT l < 1.5 * RT a && RT g < 1.5 * RT a . ( 16 ) We followed , only the subjects whose number of the smaller class trial was larger than 50 was utilized for evaluation. However, we did not balance the data as did, so that more data was available to train the network and our proposed method was able to classify unbalanced data. For DEAP, the processed data provided by the author was utilized. First, the 3 s pretrial baseline was removed from each trial. After that, the data were downsampled to 128 Hz. EOG was removed using the method described in . A band-pass filter from 4 to 45 Hz was applied. Then the average reference was conducted on the filtered data. To divide each dimension into high/low classes, five was chosen as the threshold to project the continuous nine-point scale into low and high classes in each dimension as , . Each trial was further split into 4 s shorter nonoverlapping segments to train the neural network.
C. Experiment Settings Trial-wise n-fold cross-validation for subject-specific experiments was adopted to evaluate the proposed LGGNet. In subject-specific experiments, the training and test data are all from the same subject. To avoid potential data leakage issues caused by improper random shuffling in subject-specific experiments, we adopted trial-wise instead of segment-wise For the continuous cognitive processes in the brain, such as and emotion, the adjacent data segments in one trial are highly correlated. If one randomly shuffles the segments before the training-testing split, the highly correlated segments will appear in both training and test data. Hence, a very high classification result will be observed. However, the accuracy (ACC) will drop when the highly correlated segments are never seen by the model in a real-world situation. For the attention and DEAP datasets, each trial was split into shorter segments as . The trial-wise shuffling ensures that the highly correlated segments within a trial do not appear in both train and test data in a cross-validation fold. The nested cross-validation was utilized to avoid biased evaluation. The outer loop of the nested cross-validation was the trial-wise n-fold cross-validation, and the inner loop was another k-fold cross-validation, where n DEAP = n fatigue = 10, n attention = 6, and k = 3 in this work. The mean ACC and F1 score of all subjects were reported as the final evaluation criterion as . In the inner loop, to make full use of the training data, a two-stage training strategy was utilized as well. More details about the two-stage training process are provided in the next section.
D. Two-Stage Training The optimization process via two-stage training is introduced here. To make full use of the training data, for each step of trial-wise n-fold cross-validation, the neural networks were trained in two stages using the training data. Since the inner loop of the nested cross-validation was the k-fold crossvalidation, one fold of training data was utilized as validation data in each step of the k-fold cross-validation. First, the bestperforming model in the k folds was saved as the candidate for testing. Then, all k folds of the training data were combined as the new training data. The candidate model was fine-tuned on the combined training data with a smaller learning rate compared with the first-stage training. In the second stage, the pretrained model was trained for a maximum of 20 epochs. The training process stopped when the training ACC reached 100% the first time to make sure the model was well fine-tuned without overfitting. Test data was not used in any step of the two-stage model training. After getting the fine-tuned model, it was evaluated on the test data.
E. Implementation Details The code was implemented using PyTorch library, and the source code can be found via this link. 4 Cross-entropy loss was selected as the objective function to guide the training process. For model training, the maximum training epoch of the first stage was 200 while the one for stage II was 20 instead. The batch size was 64. The dropout rate was set as 0.5 for all three datasets. Adam optimizer was utilized to optimize the training process with the initial learning rate being 1e-3 which was scaled down by a factor of 10 in the second stage. For the attention dataset, we used 1e-2 as the initial learning rate because it yielded higher validation ACC. Early stopping was applied to reduce the training time and overcome overfitting. We set the hidden size of GCN to 32 and the number of T kernels to 64 for all three datasets. We tuned the pooling size of the power layer on the attention dataset based on the performance on the validation set and applied the same value to the fatigue dataset. Note the hyperparameter settings were the same for all the subjects within each dataset. Label smoothing with a 0.1 smoothing rate was applied when training networks on DEAP dataset because the classes were highly unbalanced for some subjects. For more details, please refer to the open-access GitHub repository for LGGNet.
V. RESULTS AND DISCUSSION The performances of LGGNet were compared with CNN, RNN, and GNN-based SOTA methods in the BCI domain. The CNN-based methods include: DeepConvNet , EEG-Net , and TSception . The RNN-based methods include: R2G-STNN and HRNN . The GNN-based methods include: RGNN , AMCNN-DGCN , and GraphNet . All the methods were under the same generalized evaluation settings which were utilized to avoid data 4 https://github.com/yi-ding-cs/LGG This article has been accepted for inclusion in a future issue of this journal. Content is final as presented, with the exception of pagination. leakage issue. For fair comparisons, all the baseline methods used the optimal parameters suggested by their authors and we used the same training codes and settings as that of LGGNet. In this section, we first show the accuracies and F1 scores against the SOTA methods with statistical analysis. Extensive analysis experiments were conducted to understand LGGNet better, including ablation studies, the effect of the LGGs, and the effect of the activation function in temporal convolutional layer. Then saliency maps were utilized to visualize the most informative region of the data identified by LGGNet. The learned adjacency matrices were visualized to see what relations of the local graphs were learned by LGGNet.
A. Statistical Analysis We first report the mean ACC and mean F1 score on the three benchmark datasets for four types of cognitive tasks (shown in Table II ). The two-tailed Wilcoxon signed-rank test was utilized for the statistical analysis on the attention dataset and DEAP, while paired T -test was used on the fatigue dataset because there were fewer subjects in the fatigue dataset.
1) Attention Classification Task: LGGNet-G achieves the highest classification results in most of the experiments, especially for the attention dataset, on which the improvements in accuracies are all statistically significant. The accuracies of LGGNet-G are 9.12% ( p < 0.01), 13.28% ( p < 0.001), 9.05% ( p < 0.001), 7.69% ( p < 0.05), 6.77% ( p < 0.05), 6.77% ( p < 0.05), 6.48% ( p < 0.05), and 5.56% ( p < 0.05) higher than these of GraphNet, AMCNN-DGCN, RGNN, HRNN, R2G-STNN, TSception, EEGNet, and DeepConvNet, respectively. The improvements achieved by LGGNet-G in F1 scores over these baselines are 6.48% ( p < 0.05), 12.69% ( p < 0.01), 8.54% ( p < 0.01), 9.20% ( p < 0.05), 6.41% ( p < 0.05), 6.47% ( p < 0.05), 5.34% ( p = 0.091), and 0.10% ( p = 0.928) respectively. 2) Fatigue Classification Task: On the fatigue dataset, the best ACC and F1 score are achieved by LGGNet-F with most of the improvements being statistically significant. LGGNet-F achieves 90.76% ACC in fatigue detection tasks, which are 11.98% ( p < 0.001), 11.81% ( p < 0.001), 7.21% ( p < 0.01), 12.8% ( p < 0.05), 1.97% ( p < 0.01), 4.59% ( p < 0.01), 3.14% ( p = 0.231), and 23.9% ( p < 0.01) higher than the ones of GraphNet, AMCNN-DGCN, RGNN, HRNN, R2G-STNN, TSception, EEGNet, and DeepConvNet, respectively. The improvements in F1 scores over these baselines are 15.7% ( p, 0.05), 14.46% ( p < 0.01), 10.27% ( p < 0.05), 17.42% ( p = 0.068), 1.76% ( p < 0.05), 5.41% ( p < 0.05), 5.33% ( p = 0.302), and 15.39% ( p < 0.05), respectively.
3) Emotion Classification Task: LGGNet-G still achieves the highest F1 score (64.51%) in the emotion classification task, while the best ACC is achieved by R2G-STNN (60.11%). The differences in accuracies on the DEAP dataset are less than the ones on the other datasets, but all the LGGNet variants achieve relatively larger improvements over the baselines. Compared with GNN-based methods, LGGNet-G has 5.36% ( p < 0.001), 6.28% ( p < 0.001), and 1.29% ( p = 0.242) higher ACC than GraphNet, AMCNN-DGCN, and RGNN. And the improvements in F1 scores are 9.92% ( p < 0.001), LGGNet-G achieves higher ACCs and F1 scores than all the RNN and CNN-based baselines, except R2G-STNN.
4) Preference Classification Task: LGGNet-H achieves the highest ACC (63.07%) and F1 score (72.53%) in the preference classification task among three variants of LGGNet, while RGNN achieves the highest ACC (63.60%) and F1 score (73.90%) among all the compared methods. But the performance differences between LGGNet-H and LGGNet-G are not significant. Except for RGNN, LGGNet-H has higher ACCs and F1 scores than the compared baseline methods. Especially for GNN-based methods, LGGNet-H has 1.01% ( p = 0.271) and 1.12% ( p = 0.190) higher ACCs than GraphNet and AMCNN-DGCN. And LGGNet-H has 0.74% ( p = 0.358) and 1.59% ( p = 0.052) higher F1 scores than GraphNet and AMCNN-DGCN.
B. Ablation Study To better understand the individual contribution of the components kernel-level attentive fusion, local-graph filtering, and global-graph filtering in LGGNet, ablation studies were conducted by removing each of these blocks from the LGGNet-H network. DEAP dataset was utilized because there were more data and subjects compared to the other datasets. The ablation studies were conducted on the preference classification task because the performances were better than the ones for the emotion classification task using DEAP. Hence, LGGNet-H was utilized because it achieved the best classification results among the proposed methods. In the first ablation study, to investigate the contribution of the kernel-level attentive fusion, this block was removed from the network and the output of the temporal convolutional layer was reshaped as node by feature and was sent directly to the local-graph filtering layer. In the second study, the learned temporal representations from the temporal learning block were used as the input of the global-graph filtering layer directly to obtain performance without local-graph filtering. Finally, the feature output from the local-graph filtering layer was flattened and passed to the MLP to get the output of network without global-graph filtering. The new classification accuracies and the performance changes are reflected in Table III score, it even drops more with the decrease being 4.08%. The results show the effectiveness of the kernel-level attentive fusion.
2) Contribution of the Local-Graph Filtering: To understand the contribution of the local-graph-filtering layer, it was removed from LGGNet. In this case, each EEG channel is one node in the graph and the global adjacency matrix, A global ∈ R c×c , reflects the connection among all the nodes (c is the number of EEG electrodes). According to the results shown in the second row of Table III , after removing the local-graph-filtering layer entirely, the ACC drops from 63.07% to 60.93%, decreasing by 2.14%. For the F1 score, it drops by 3.17%. This indicates the importance of the local-graph-filtering layer. 3) Contribution of the Global-Graph Filtering: The globalgraph filtering was removed from the LGGNet to analyze its importance to the classification performance. In this situation, only the local-graph-filtering layer is kept to learn the spatial pattern of EEG. After getting the embeddings of local graphs, the latent representation was fed into fully connected layers without global-graph filtering. According to the third row of Table III , the ACC and F1 score all drop after removing the global-graph-filtering layer. And the decreases are higher than the ones without the localgraph-filtering layer. A 4.01% drop was observed for ACC after discarding the global-graph-filtering layer, while the one for the F1 score was 5.45%. The results show the contribution of the global-graph filtering is larger than the one of localgraph filtering in LGGNet.
C. Effects of Activation Functions in the Temporal Convolutional Layer To study the effects of different activation functions, we replaced the power layer with commonly used activation functions, such as ReLU, leaky-ReLU, exponential linear unit (ELU), and scaled ELU (SELU) separately. Replacing the power layer with other commonly used activation functions causes the decrease of the classification results. The results are shown in Table IV . Using leaky-ReLU() in the temporal convolutional layer has the least drops in ACC (1.43%) and F1 score (2.18%). The largest drops were observed when we replaced the power layer with SELU() activation function, which were 2.09% in terms of ACC and 3.14% in terms of F1 score. This indicates the importance of the power layer.
D. Effects of LGGs To evaluate the effects of treating EEG as LGGs that are specially designed according to neuroscience, we compare them with a none LGG baseline. Only global-graph convolution was conducted because there were no local graphs in the baseline. The effects of different LGG definitions were also analyzed by comparing their performances on different cognitive tasks. The results are shown in Figs. 3 and 4 . And the detailed accuracies and F1 scores of three LGGNet variants are shown in the last three rows of Table II . Using LGGs that are specially designed according to neuroscientific evidence yields significant improvements on classification performances for all four cognitive tasks, except the ones for the attention classification task when frontal and hemisphere LGGs are used in LGGNet. Compared with the baseline, LGGNet-G achieves 2.63% ( p = 0.087) and 4.50% ( p < 0.05) higher ACC and F1 score than those of the baseline for the attention classification. For the fatigue detection task, the improvements achieved by using LGGNet-G are 2.99% ( p < 0.01) and 2.75% ( p < 0.01). In the emotion classification task, a 2.49% ( p < 0.001) higher ACC and a 4.48% ( p < 0.001) higher F1 score are observed when the general LGG is used than the ones of the baseline that uses the none LGG. The improvements achieved by LGGNet-G on the preference classification task are 2.26% ( p < 0.01) and 3.33% ( p < 0.001) in terms of ACC and F1 score. The results indicate the effectiveness of using LGGs to extract the spatial information of EEG. The general LGG has a higher generalization ability as expected. LGGNet using the G g achieves the highest classification accuracies and F1 scores for both attention and emotion classification tasks. However, in the mental fatigue classification task, LGGNet-F achieves the highest F1 score and the highest ACC. LGGNet-H achieves the highest classification results for the preference classification task instead. But the differences in performance are not significant for fatigue, emotion, and preference classification tasks. This suggests adding more symmetric local graphs in functional areas can yield certain improvements over the general LGG for some tasks but the improvements are not significant.
E. Interpretability and Visualization 1) Saliency Maps Visualization: In this section, the saliency map is utilized to visualize which parts of the data are more informative. To better visualize the saliency map, the original saliency map was averaged along the time dimension to get the topological map of the EEG channels for each subject. Fig. 5 shows the averaged saliency map of all the subjects. From Fig. 5 (a), LGGNet mainly learns from temporal (T7 and T8), and parietal area (POz) of the brain for attention classification. This is also suggested in and that the temporal and parietal lobes are attention-related regions. The frontal area provides more fatigue classification-related information to LGGNet. According to Fig. 5 (b), strong activations are observed on Fp1, Fp2, F7, FC3, and FC4. This is consistent with other studies which indicate the frontal lobe is related to human fatigue states. From Fig. 5(c), LGGNet learns more emotional information from the temporal (T7 and T8) and frontal (Fp2, AF4, and FC2) areas of the brain. Temporal area, especially the left temporal area provides more emotion related information, which is in line with . Frontal area also contributes to the final classification results. This is consistent to and , which indicate that the frontal area is related to emotions. According to Fig. 5(d), LGGNet learns more from the temporal areas (T7 and T8) of the brain. Neuroimaging study suggests the temporal lobes are predictive for the preference prediction during video watching, and these brain areas are related to sensory integration and emotional processing. The above neurological knowledge indicates the neural network learns from the task-related regions of EEG signals. 2) Learned Global Connection Visualization: In this section, the final learned adjacency matrices and learnable attentive masks of each task's best performing model are visualized in Figs. 6
and 7 to understand what relations LGGNet learns from EEG for different cognitive tasks. To get a general view of each cognitive task, the normalized learnable attentive masks and adjacency matrices were averaged for all the subjects. All the negative values in the learnable attentive masks were set zero before normalization because of the ReLU activation function in (12) . Because the adjacent relations are among local graphs instead of individual EEG channels, the names of the local graphs are defined by the name of the functional area. The 'l' and 'r' are utilized to indicate the location of the symmetric subgraphs within a functional area. a) Attention: Some connections between frontal and parietal regions, between 1) AF and PO and 2) FC and PO, are observed in the adjacency matrix shown in Fig. 6(a) for attention classification task. And for the self-connections, frontal (Fp), parietal (CP and P), and temporal (T-l and T-r) have higher attentive weights. We further visualize the learned attentive mask to see the learned relations among different local areas. According to Fig. 7 (a), some connections between frontal and parietal are enhanced by the attentive mask. Besides, the self-loops of CP and T-l get more attention weights. It is consistent with which indicates the posterior parietal lobe (PPL) that has dense connectivity with the cortical and subcortical regions in frontal, temporal, and occipital lobes. b) Fatigue: According to Fig. 6 (b), relatively stronger connections are observed among frontal subareas (between Fp-r and F-l) for the fatigue classification task. The connections between frontal and occipital areas, frontal and motor areas are also relatively strong. These connections are between 1) Fp-r and O, 2) F-l and CP, 3) Fp-l and C, and 4) C and Z. For the self-connections, more attentive weights are given to frontal (Fp-l, F-l, and FC-l), parietal (P), and occipital (O). The frontal lobe and parietal areas are related to mental attention functions . The relatively strong connections among frontal, occipital, and motor areas may be because the visual and motor processes were involved in the fatigue experiment (driving in VR). For the learned attentive mask, according to Fig. 7 (b), some connections between frontal and occipital are enhanced by the attentive mask. For the selfloops, the one of area O has the highest attention weight. c) Emotion: More connections among frontal, occipital, and temporal, which are between 1) AF and FC, 2) AF and O, 3) CP and O, and 4) P and T-r, are observed in the final learned adjacency matrix [Fig. 6(c) ] than the ones for the attention and preference classification tasks. It is also consistent with neuroscience that the emotional process involves more basic processes, such as perception and attention. For selfconnections, the frontal and temporal areas, commonly known as the emotion-related areas, have higher attention weights than the others. However, C, CP, and P also get some attention More attentive weights are given to the temporal area (T-l, T-r) than the other regions. But the frontal (AF-r, F-r, FC-r) and occipital (PO-l and PO-r) areas are also highlighted. For the attentive mask shown in Fig. 7 (d), the connections that are enhanced by the attentive mask are between 1) F-l and AF-r, 2) P-r and F-l, 3) PO-r and C-r, and 4) PO-r and CP-r. Among self-loops, F-r, CP-l, P-l, PO-l, T-l, and T-r get higher attention weights than the others. Visualizing the learned adjacency matrices and the learnable attentive masks shows the relations and the important local regions identified by LGGNet. And most of the learned relations are task-related. Since the cognitive processes are complex and may involve more basic processes that are not unique to the task, more analysis should be conducted in the future to better understand what and how the network learns from EEG. Although LGGNet achieves the highest classification results for most of the experiments for four cognitive tasks, the limitation of this work should also be noticed. In this work, the nodes within each local area are set to be fully connected, which might not be able to reflect the complex brain activities inside that functional area. How to model the relations within local areas should be explored. The learned connections in the adjacency matrices of attention, emotion, and preference task are not as strong as the ones of fatigue task. Further improvement of the network or loss function design should be considered in the future to improve the classification performance.
VI. CONCLUSION In this article, we propose LGGNet, a neurologically inspired GNN, to learn from LGG representations of EEG. Multiscale 1-D temporal convolutional kernels with kernellevel attention fusion are utilized to learn the temporal dynamics of EEG. Local-and global-graph filtering learns the brain activities within each functional area and the complex relations among them during the cognitive process in the brain, respectively. With a robust nested cross-validation strategy, the proposed method and several SOTA methods are evaluated on three publicly available benchmark datasets for attention, fatigue, emotion, and preference classification tasks. The proposed method achieves significantly ( p < 0.05) higher accuracies and F1 scores than other methods in most of the experiments. Further analyses also show that applying neuropsychological knowledge to the network design ensures that networks are trained on task-specific neural activations. Fig. 1 . 1 Fig. 1. Structure of LGGNet. LGGNet has two main functional blocks: the temporal learning block and the graph learning block. The temporal convolutional layer and the kernel-level attentive fusion layer are shown in the (a) temporal learning block. The local-and global-graph-filtering layers are shown in the (b) graph learning block. The temporal convolutional layer aims to learn dynamic temporal representations from EEG directly instead of human extracted features. The kernel-level attentive fusion layer will fuse the information learned by different temporal kernels to increase the learning capacity of LGGNet. The local-graph-filtering layer learns the brain activities within each local region. Then the global-graph-filtering layer with a trainable adjacency matrix will be applied to learn complex relations among different local regions. Four local graphs are shown in the figure for illustration purposes only, the detailed LGG definitions are provided in "Defining LGGs of EEG" of Section III-B. Best viewed in color.
Fig. 2 . 2 Fig. 2. Three types of LGG definitions. (a) General LGG definition. This local-graph structure is defined according to the 10-20 system. Each local graph reflects the brain activities of a certain brain functional area. (b) Frontal LGG definition. Based on the general LGG, the neuroscience evidence of frontal asymmetry patterns in frontal areas is further considered. Six frontal local graphs that are symmetrically located on the left and right frontal areas of the brain are added to learn more discriminative information. (c) Hemisphere LGG definition. The symmetrical local graphs are added for all the functional areas defined in the general LGG. The nodes in a local graph are in the same color. The dotted lines are the local graphs. This diagram illustrates the definition for the 62 channel EEG.
Fig. 4 . 4 Fig. 4. Mean F1 scores of LGGNet using different graph structures. The blue bar is the baseline that has no LGGs.
Fig. 5 . 5 Fig. 5. Mean saliency maps of all subjects for three datasets. These mean saliency maps are for: (a) attention; (b) fatigue; (c) emotion; and (d) preference.
Fig. 6 . 6 Fig. 6. Visualization of the final learned adjacency matrices for four cognitive tasks. These mean adjacency matrices are for: (a) attention; (b) fatigue; (c) emotion; and (d) preference.
Fig. 7 . 7 Fig. 7. Visualization of the learnable attentive masks for four cognitive tasks. These mean attentive masks are for: (a) attention; (b) fatigue; (c) emotion; and (d) preference.
Concatenation of tensors.G g ,G f , G h General, frontal, and hemisphere graphs. l Length of the EEG sample in time dimension. () Activation functions. Z Output tensor of a neural network layer. t Number of temporal (T ) kernels. f Length of features. F () Operations. () A local Local adjacency matrix. W Trainable weight matrix. b Trainable bias vector. Hadamard product. r, R Index and total number of local graphs. p, P Index and number of nodes in a local graph. h local Latent representations of local graphs. A global Global adjacency matrix. Dot product. M Trainable attentive mask of global adjacency matrix. w Trainable weight. D Degree matrix of the adjacency matrix. h
is the Hadamard product. After local-graph filtering, the attentively filtered representation within each local graph will be aggregated by an aggregating function F aggregate () to get the hidden embeddings of the local graphs. Let Z i R ] T be the locally filtered graph representations, where Z r ∈ R P r × f is the local-graph representation, R is the total number of local graphs, P r is the number of nodes in the r th local graph ( P r = c), and f is the feature length of each node after local-graph filtering. A local graph can be denoted as: The aggregating function aggregates the node vectors within each local graph. It can be maximum, minimum, average, etc. In LGGNet, the average operation is selected as the aggregating function. Hence, the output of the local-graph-filtering layer, Z i Z r = [z 1 r , . . . , z p r , . . . , z P r r ] T , where z p r is the node vector in the local graph. filtered = [Z 1 , . . . , Z r , . . . , Z local ∈ R R× f , can be calculated by
ground truth label y; graph definitions G g , G f , and G h ; global adjacency matrix A global Output: pr ed, the prediction of LGGNet do local filtering on each node attribute by Eq. 8; 9 aggregate the filtered node attribute within each local graph (G g , G f , or G h ) by Eq. 9; 1 Initialization; 2 for j ← 1 to 3 do 3 get j th temporal kernel size by Eq. 1; 4 get Z temporal by Eq. 2 using X i as input; j 5 end 6 get Z i T -M S by Eq. 3; 7 do kernel-level attention fusion by Eq. 4 to get Z i f use ; 8 10 get the A global by Eq. 10 -13; 11 do global filtering on embeddings of local graphs by Eq. 14 with A global ; 12 get pr ed by Eq. 15;
TABLE II COMPARISON II BETWEEN OUR PROPOSED LGGNET AGAINST SOTA CLASSIFIERS ON THREE BENCHMARK DATASETS USING TRIAL-WISE n-FOLD CROSS-VALIDATION
TABLE III RESULTS III OF ABLATION STUDIES ON DEAP USINGLGGNET-H 9.00% ( p < 0.001), 3.23% ( p = 0.126).
.1) Contribution of the Kernel-Level Attentive Fusion:According to the results shown in the first row of TableIII, removing the kernel-level attentive fusion makes the ACC drop from 63.07% to 60.95%, decreasing by 2.12%. For the F1
TABLE IV EFFECT IV OF THE ACTIVATION FUNCTION IN THE TEMPORAL CONVOLUTIONAL LAYER OF LGGNET-H ON DEAP Fig. 3. Mean accuracies of LGGNet using different graph structures. The blue bar is the baseline that has no LGGs.
This article has been accepted for inclusion in a future issue of this journal. Content is final as presented, with the exception of pagination.
|
10.4314/as.v17i2.4
|
cc-by
| null | null |
openalex
|
Okra (Abelmoschus esculentus), pepper (Capsicum spp.) and tomato (Solanum lycopersicum) are rich sources of minerals and vitamins required by humans for normal growth and development. However, the productivity of these vegetables is seriously constrained by Cucumber mosaic virus (CMV) disease. To date, adoption of resistant cultivars is the most effective, ecologically sound and sustainable management strategy against the disease. Therefore, selected cultivars of okra (Clemson Spineless, Ex Bassawa -2 and NHAe 47 -4), pepper (California Wonder, Cayenne and Yolo Wonder) and tomato (Roma Savanna, Tropimech, and UC82B) were evaluated for resistance to CMV. The experiment was conducted under screenhouse and field conditions in Minna, Southern Guinea Savanna zone of Nigeria. Completely randomised design with six replications was used in screenhouse while the field trial was laid out using randomised complete block design with three replications. Seedlings were inoculated with CMV at 10 days after emergence. Disease incidence, disease severity, growth and yield characteristics were recorded. Data were subjected to analysis of variance at p <= 0.05. Infected plants elicited mosaic symptoms but disease severity was cultivar dependent. Plant height, leaf area, fruit length, fruit diameter and fruit weight were all reduced by CMV. The lowest disease incidence ( < 35 %), severity ( < 3), reductions in morphological and yield parameters ( < 10 %) were found in Ex-Bassawa-2, Cayenne, and Roma Savanna. This indicated that they contained CMV tolerant genes. Therefore, the three most tolerant cultivars (Ex-Bassawa-2 cultivar of okra, Cayenne of pepper and Roma Savanna of tomato) are recommended to farmers in CMV prone areas.INTRODUCTION Vegetables have been recognized as important components of human nutrition particularly in sub-Saharan Africa (SSA) where there is a serious problem of malnutrition (Abro and Sadaqat, 2010) . These crop species are not only good sources of essential vitamins and minerals (Bakhru, 2003) , they are widely used as the complement to starchy staple foods. In Africa, vegetables constitute the fourth largest group of commodities produced for various uses. Okra (Abelmoschus esculentus L. Moench) also known as lady's finger is a member of the family Malvaceae (Walker, 2012) . It is cultivated in several parts of tropical and sub-tropical Africa (Saifullah and Rabbani, 2009) . Okra is grown on subsistence and large-scale farms in Iran, Turkey, West Africa, many Asian countries and the southern United States. In 2016, the world total okra output was approximately 8.9 million tonnes. India was the largest producer with about 5.5 million tonnes, followed by Nigeria which produced approximately 2 million tonnes and Sudan had about 287, 300 tonnes (FAO, 2016) . Pepper (Capsicum spp.), a vegetable of global importance is believed to have originated in Central and South America. Its cultivation later spread to the New World such as Thailand, Indonesia, India, Malaysia and Philippines in Asia; and Nigeria, Ghana, Senegal, and Kenya in Africa in 1943 (Rehima, 2006) . It is a dependable source of income for smallholder farmers in developing countries such as Bhutan, Cambodia, China, Ethiopia, Ghana, India, Indonesia, Nigeria, Pakistan and Thailand (Lin et al., 2013) . Today, hot pepper (chili) is an important commodity in world spice trade and the popularity of sweet pepper has continued to rise in the tropics. Peppers can be consumed in fresh, dried or processed form (Idowu-Agida et al., 2010) . In 2016, the world production of peppers was estimated at 546.3 million tonnes. Nigeria was the largest producer in Africa with about 67, 000 tonnes (FAO, 2016) . Tomato (Solanum lycopersicum) is an herbaceous fruiting plant (Babalola et al., 2010) . Though believed to have originated in Latin America, it has been identified as one of the most widely grown vegetables with the ability to survive in diverse environmental conditions (Rice et al., 1987) . Tomato fruit is rich in vitamins and minerals that are essential in the human diet. It may be eaten fresh as salad or processed into purees or pastes which are basically used for stews and soups as well as fruit drinks (Babalola et al., 2010) . The world tomato production in 2016 stood at 177 million tonnes (FAO, 2016) . In Africa, Egypt with about 7.9 million tonnes was the largest producer, followed by Nigeria which produced approximately 2.2 million tonnes. In spite of the numerous uses of okra, pepper, and tomato, their productivity is adversely constrained by insect pests and diseases (Feldmann et al., 2009) . Vegetable crops are attacked by several pathogens including Cucumber mosaic virus (CMV). Cucumber mosaic virus is pathogenic on over 1000 plant species in different genera and families (Palukaitis and Garcia-Arenal, 2003) . Epidemiological studies have revealed that the virus is seed-borne (O'keefe et al., 2007) and long distance dissemination is facilitated by over 75 species of aphids in a non-persistent manner. Although insecticides may be applied to control the aphid vectors of the virus (Hartz and Cantwell, 2008) , cultivation of resistant genotypes is the most effective and sustainable approach (Salaudeen, 2016) . Adoption of virus-resistant varieties is cheap, effective, easy to adopt, environment-friendly and sustainable. A lot of vegetables, particularly okra, pepper, and tomato are grown in Minna, Southern Guinea Savanna agro-ecology of Nigeria. However, there is a scarcity of information on their level of resistance to CMV disease. It was therefore important to assess the resistance status of some of the most common cultivars of these vegetables in case of sudden CMV disease outbreak. This information would be useful for developing preventive measures against poor yield and total crop loss. Therefore, this research was conducted to determine the level of resistance in the selected vegetable cultivars to Cucumber mosaic virus disease.
MATERIALS AND METHODS
Virus source and maintenance An isolate of CMV obtained from the stock in the Department of Crop Production, Federal University of Technology, Minna, Nigeria was used for inoculation. The isolate was maintained at room temperature over silica gels in vial bottle. The CMV inoculum was multiplied in the plants of a susceptible cowpea cultivar, Ife Brown. This was accomplished by grinding the CMV-infected leaves in extraction buffer, pH 7.2 (0.1M sodium phosphate dibasic, 0.1M potassium phosphate monobasic, 0.01M ethylene diamine tetra acetic acid and 0.001M L-cysteine per litre of distilled water) at the ratio of 1:1 w/v (1 g leaf in 1 mL inoculation buffer). Just before inoculation, the upper leaf surface was dusted with carborundum powder (600-mesh) (Fisher Scientific, Fair Lawn, NJ) . A piece of cheesecloth was dipped in the virus extract and then rubbed on the dusted leaf surface. At three weeks after inoculation symptomatic leaves were harvested from infected plants and subjected to Enzyme-Linked Immunosorbent Assay (ELISA), using CMV polyclonal antibody (Kumar, 2009) . The ELISA positive leaf tissues were preserved on anhydrous calcium chloride (CaCl2) in plastic vials by placing 10 g of the salt in the vial covered with a thin layer of nonabsorbent cotton wool and 1 g of the virus-infected leaf was folded into the vial and covered. The vials were maintained at room temperature (37 oC).
Source of seeds The three most popular okra (Clemson Spineless, Ex-Basawa-2 and NHAe47-4), pepper (California Wonder, Cayenne and Yolo Wonder), and tomato (Roma Savanna, Tropimech and UC82B) cultivars in the study area were evaluated. Seeds of Ex-Basawa-2 and NHAe 47-4 were respectively sourced from the Institute for Agricultural Research (IAR), Samaru, Ahmadu Bello University, Zaria, Kaduna State, Nigeria and from the stock in the Department of Crop Production, Federal University of Technology, Minna. Clemson Spineless okra, pepper and tomato seeds were purchased from an Agrochemical store in Minna. These cultivars were selected because they are the most popular vegetables in the study area.
Screenhouse Study Crop establishment and inoculation The screenhouse experiment was conducted under screenhouse conditions at the Teaching and Research Farm, Federal University of Technology, Minna, Niger State during the 2014 cropping season. It was carried out using three independent trials. The three cultivars of each crop constituted the treatments. Treatments were arranged as Completely Randomized Design (CRD) with six replications. This arrangement was made for both inoculated and uninoculated (control) treatments of each cultivar. Seeds of the selected okra, pepper and tomato cultivars were sown in plastic pots (30 cm diameter and 30 cm high). The pots were perforated at the bottom and then a thin layer of small stones was placed in them, followed by a thin layer of dry grass to ensure good drainage. The pots were then filled with heat-sterilized topsoil (3.8 kg per pot) and placed on iron benches (8 m × 12 m) in a screenhouse. Grooves not more than 1.5 cm deep and 7.5 cm apart were made and the okra seeds were dispensed into them. The tomato and pepper seeds were sown in drills and firmed with soil. The soil was then sprinkled with water from a watering can. At 10 days after emergence, seedlings were thinned to one plant per stand, maintaining five plants per pot. All seedlings for virus trial were inoculated with CMV extract at 10 days after emergence. At the time of inoculation, CMV inoculum was recovered from the dehydrated leaf tissues by grinding in extraction buffer, pH 7.2. The procedure for inoculation was as described above. Uninoculated plants of each cultivar were kept in a separate screenhouse to serve as the control.
Field Study Experimental site Field evaluation was conducted at the Teaching and Research Farm of the Federal University of Technology, Minna, Niger State, Nigeria, during the 2014 cropping season. The site used for the experiment has been under the cultivation of arable crops (maize, cowpea and yam) in the last five years. The bearing and elevation of the site (90 51'N, 60 44'E; 212 m above sea level) were captured using the Geographical Positioning System (GPS, Garmin, Taiwan) equipment.
Crop establishment, management and inoculation The site with a field size of 649 m2 was cleared of the existing trash. The field was sprayed with a systemic herbicide (glyphosate) at the rate of 3 litres ha-1 on 13th July 2014. This was followed by ridging on 28th July 2014. Seeds of the three vegetable cultivars were sown on 31st July 2014. They were sown directly on ridges at a spacing of 75 cm (interrow) and 50 cm (intra-row). Mulching was done using dry grass to prevent raindrop splashes, conserve soil moisture and also to prevent birds from picking out already sown seeds. Seedlings were thinned to one plant per stand at 1 week after emergence. Seedlings were inoculated with CMV extract at 10 days after emergence, using the same procedure described for the screenhouse evaluation. Both inoculated and uninoculated plants were sprayed with insecticide (Cypermethrin 10 % E.C.) at the rate of 0.5 litre ha-1 at 2-week interval. The first weeding was done manually with a hoe at 14 days after planting. Subsequent weedings were carried out manually at two and four weeks intervals. Fruits were harvested manually with pruning scissors for okra and with hands for pepper and tomato using hand gloves. This was carried out at an interval of four days when the vegetables were fully matured, that is when the tip of okra fruits were observed to break easily when snapped with fingertips and as the pepper and tomato fruits turned red.
Treatments and experimental design Treatments consisted of three cultivars of okra (Clemson Spineless, Ex Bassawa -2 and NHAe 47-4), pepper (California Wonder, Cayenne and Yolo Wonder) and tomato (Roma Savanna, Tropimech and UC82B). Three independent trials were laid out for okra, pepper and tomato cultivars in a Randomized Complete Block Design (RCBD) with three replications. Each treatment was evaluated in four ridges (each 5 m long) with 0.75 m interrow spacing for both inoculated and uninoculated (control) plots.
Data collection and statistical analysis The plants were assessed for disease incidence, disease severity, morphological and yield characters. Disease incidence was taken as a percentage of the inoculated plants exhibiting symptoms of infection at 1 and 2 weeks after inoculation (WAI). Disease severity was recorded as symptom severity scores (SSS), using 1 -5 visual scale (Arif and Hassan, 2002) . On the scale, 1 -no symptoms (apparently healthy plant); 2 -slightly mosaic leaves (10 -30 %); 3 -mosaic (31-50 %) and leaf distortion; 4 -severe mosaic (51-70 %), leaf distortion and stunting; 5 -severe mosaic (>70%), stunting and death of plants. Percentage reduction/loss (%) was calculated for each of the growth and yield parameters (plant height, leaf area, fruit length, fruit diameter and fruit weight) using the formula: Reduction/loss (%) × 100 (Sarra, 2005) where: Xu -Mean of uninoculated plants Xi -Mean of inoculated plants Data were subjected to Analysis of Variance using the PROC GLM of SAS (Statistical Analysis System, 2008) . Where F test was significant, means were separated using the Least Significant Difference (LSD).
RESULTS
Cucumber mosaic virus disease incidence The data on CMV disease incidence from the evaluated vegetables are presented in Table 1 . In both screenhouse and field trials, symptoms of CMV infection were first observed at five days after inoculation (DAI) for pepper and 6 DAI for okra and tomato, except for Cayenne, Ex-Bassawa-2 and Roma Savanna in which first symptoms were observed at 7 DAI, 10 DAI and 14 DAI, respectively. Symptoms observed included leaf discolouration, followed by mosaic on leaves and stunted growth on infected Clemson Spineless and NHAe 47-4 cultivars of okra. On pepper cultivars, symptoms observed were leaf discolouration followed by mild mottling of leaves to severe mosaic on leaves and stunted growth, particularly in Yolo Wonder. Chlorosis, severe mosaic and stunted growth were also observed in infected plants of California Wonder. Conversely, Cayenne plants elicited very mild leaf mottling. Symptoms observed in infected tomato plants included chlorosis of the older leaves, blistering and stunted growth. At 1 WAI, there were no symptoms on Roma Savanna but at 2 WAI, mild mosaic symptom was observed. There was no apparent symptom of infection on the leaves of uninoculated (control) plants. Disease incidence differed significantly (p<0.05) among the crop cultivars. At 1 WAI, the highest disease incidence was observed in infected plants of Yolo Wonder (82.5 %) while the inoculated plants of Roma Savanna did not exhibit disease symptom. Among the okra cultivars, disease incidence was significantly highest in infected plants of NHAe47-4 (37.5 %), followed by Clemson Spineless (28.3 %) while the lowest was found in Ex-Bassawa-2 (10 %). In pepper, the diseased plants of Yolo Wonder suffered the highest incidence of infection (82.5 %) while Cayenne exhibited the lowest level of infection (20 %). In tomato, the highest disease incidence was observed in UC82B (30.8 %), followed by Tropimech (25 %) while the inoculated plants of Roma Savanna were apparently symptomless. At 2 WAI, although the incidence of CMV infection was highest in NHAe47-4 (95.8 %), the value observed was not significantly different from that in Clemson Spineless (90.8 %). In the field trial, disease incidence was still significantly lower in Clemson Spineless than NHAe47-4. Conversely, disease incidence was mildest in infected plants of Ex-Bassawa-2 at 1 WAI (16.7 %) and 2 WAI (22 %). Among the pepper cultivars, disease incidence at 2 WAI took the same trend as observed at 1 WAI (28.3-100 %) but higher values were obtained at 2 WAI (31.3 -100 %). In tomato plants, the trend of infection at 1 WAI (0-30.6 %) was similar to that observed at 2 WAI (18.1 -81.7 %).
Cucumber mosaic virus disease severity The severity of CMV infection varied significantly among crop cultivars as shown in Table 2 . In both screen house and field trials, symptom expression was generally low at 2 WAI except in Yolo Wonder and California Wonder which showed intermediate to high level of infection. Among the okra cultivars, disease severity was consistently highest in NHAe 47-4 (score -2.8-4.1), followed by Clemson Spineless (score -2.4-3.9), while Ex-Bassawa-2 (score -1.1-1.9) consistently elicited the lowest symptom severity. For pepper, the lowest disease severity was observed in inoculated plants of Cayenne (score -1.5-2.9) throughout the period of evaluation. A similar trend was observed among the tomato cultivars in which Roma Savanna (score -1.1-1.7) consistently expressed the lowest disease severity symptoms. Although disease severity was consistently highest in infected plants of UC82B (score -1.8-3.4), symptom scores were not as dramatic as in NHAe 47-4 (score -2.8 -4.1), Yolo Wonder (score -3.4-5) and California Wonder (score -3.1-4.2).
Effect of cucumber mosaic virus disease on plant height Uninoculated plants of all the evaluated cultivars exhibited normal and rapid growth contrary to those infected with CMV. The virus restricted plant height at varying levels among the crop cultivars (Table 3 ). For the screenhouse trial, uninoculated plants were significantly taller than the CMV infected plants except for Ex-Bassawa-2, Cayenne and Roma Savanna. However, in the field trial, uninoculated plants of all the cultivars were significantly taller than their infected counterparts. Among the three vegetables, height reduction was markedly highest in Yolo Wonder in both screenhouse (65.8 %) and field (100 %) trials. It was also observed in the field study that the inoculated plants of Yolo Wonder died as a result of CMV infection. Among the okra cultivars, height reduction was mildest in the infected plants of Ex-Bassawa-2 in both field (1.5 %) and screenhouse (1.6 %) evaluations. Conversely, while the CMV infected plants of NHAe47-4 exhibited the highest height reduction in the screenhouse study (40.6 %), field data indicated that plant height was severely impaired in the infected plants of Clemson Spineless (38.6 %). Among the pepper cultivars, the CMV inoculated plants of Cayenne exhibited the lowest height reduction in both screenhouse (6.5 %) and field (0.8 %) trials. Similarly, the CMV infected plants of Roma Savanna showed the lowest height reduction (3.4 %) while height reduction was most conspicuous in the infected plants of UC82B (45 %).
Effect of cucumber mosaic virus disease on leaf area The healthy plants produced broad leaves with normal shape while the leaves of CMV inoculated plants were narrow and twisted. The effect of CMV on leaf area of the evaluated vegetables is shown in Table 4 . The difference in leaf area between healthy and infected plants was not significant in Ex-Bassawa-2 and Roma Savanna in both screenhouse and field trials. A similar phenomenon was encountered in Cayenne under screenhouse study. Conversely, healthy plants of the remaining cultivars produced significantly broader leaves than their infected counterparts. Overall, leaf area reduction was most conspicuous in infected plants of Yolo Wonder (100 %). Reduction in leaf area was less than 1 % in infected plants of Ex-Bassawa-2 under screenhouse (0.8 %) and field (0.7 %) conditions, as well as in Cayenne under field trial (0.8 %). Low reduction in leaf area was also observed in Roma Savanna under screenhouse (7.6 %) and field (8.7 %) conditions.
Effect of cucumber mosaic virus disease on fruit length Infection by CMV reduced fruit length of the CMV infected plants while the healthy plants produced appreciably long fruits with normal shape. The data on fruit length from healthy and CMV inoculated plants are presented in Table 5 . In Ex-Bassawa-2, Cayenne and Roma Savanna, there was no significant difference in fruit length between healthy and CMV infected plants for both screenhouse and field study, as well as in Clemson Spineless under screenhouse trial. In contrast, healthy plants of the remaining cultivars produced significantly longer fruits than the infected plants. Generally, reduction in fruit length was less than 5 % in Ex-Bassawa-2 under screenhouse (3.6 %) and field trials (3.2 %). Reduction in fruit length of Cayenne and Roma Savanna was 3.4 and 2.6 %, respectively under screenhouse study. Among the evaluated vegetables, high reduction in fruit length was found in NHAe47-4 (56.4 and 60.3 %) and Yolo Wonder (53.8 and 100 %) under screenhouse and field trials.
Effect of cucumber mosaic virus disease on fruit diameter The trend in fruit diameter was somewhat similar to that reported for fruit length. Fruits of the CMV infected plants were mostly smallsized, narrow and curled, contrary to the big fruits with normal shape obtained from the healthy plants. Under screenhouse and field conditions, fruits of the healthy plants were significantly wider than infected plants except in Ex-Bassawa -2, Cayenne and Roma Savanna (Table 6 ). Generally, Yolo Wonder suffered the highest reduction in fruit diameter under screenhouse (51.1 %) and field (100 %) trials. Among the okra, pepper, and tomato cultivars, reduction in fruit diameter was mildest in Ex-Bassawa-2 (1.2 %), Cayenne (8.3 %), and Roma Savanna. (3.1 %), respectively.
Effect of cucumber mosaic virus disease on fruit weight Similar to the data on fruit diameter, the difference in fruit weight between healthy and infected plants was not significant in Ex-Bassawa-2, Cayenne and Roma Savanna under screenhouse and field trials (Table 7 ). In the remaining cultivars, however, fruits of the healthy plants were significantly heavier than the CMV infected plants. Reduction in fruit weight was highest in Yolo Wonder both in the screenhouse (94.9 %) and field (100 %) evaluation. Under screenhouse evaluation, the lowest reduction in fruit weight was observed in Roma Savanna (2.3 %) whereas Ex Bassawa-2 showed the mildest reduction in fruit weight (0.2 %) under field conditions. The fruit weight reduction observed in Roma Savanna (1 %) under field study was also negligible.
DISCUSSION All the okra, pepper and tomato plants inoculated with CMV showed symptoms of infection. This is in agreement with the finding of Appiah et al. (2014) that CMV was pathogenic on several vegetable plants. The highest level of disease incidence in Yolo Wonder is an indication of its vulnerability to CMV. The high incidence of CMV observed in this study is in agreement with Arogundade et al. (2012) who encountered up to 87 % incidence of CMV on pepper during a survey in Nigeria. Roma Savanna was the most tolerant because CMV inoculated plants were apparently symptomless till 14 days after inoculation. This observation is consistent with the findings of Gergerich and Dolja (2006) who stated that susceptibility or resistance to virus infection is determined by plant genetic makeup. The early appearance of symptoms in some cultivars corroborates the findings of Salaudeen and Aguguom (2014) when some cowpea accessions were infected with CMV. Furthermore, the symptoms observed on CMV inoculated plants were similar to those reported by Agrios (2005) where the older leaves of CMV-infected cucumber developed chlorosis and necrotic areas along the margins. Disease severity varied among the inoculated crop cultivars due to their different genetic make-up. The severity of infection increased over time in some cultivars, which is in agreement with the report of Aliyu et al. (2012) when some cowpeas were inoculated with B1CMV. Roma Savanna cultivar of tomato, which had the lowest level of CMV incidence and also elicited the mildest symptoms of infection, was probably due to the presence of CMV tolerance genes. It was observed that okra cv. Ex-Bassawa-2, pepper cv. Cayenne and tomato cv. Roma Savanna exhibited the lowest level of CMV incidence and severity in each crop group, NHAe 47-4, Yolo Wonder and UC82B cultivar of okra, pepper and tomato, respectively, exhibited the highest level of infection. This indicates variability in their tolerance to CMV infection. The observation that inoculated plants of Yolo Wonder suffered the highest level of infection (total crop loss) revealed that the genotype lacked CMV resistant genes. This finding corroborates that of Biswas et al. (2005) where the maximum incidence of disease (up to 80 %) and severity (up to 5) was observed in diseased chilli pepper. Jin et al. (2009) also reported that viral infection caused symptoms such as various forms of mosaic and distortions in plants with consequent reductions in crop growth and yield. The differences in morphological and yield data of the control (healthy) and inoculated plants are indications of pathogenicity of CMV on the tested vegetables. The okra cv. Ex-Bassawa-2, pepper cv. Cayenne and tomato cv. Roma Savanna in which reductions in plant height, leaf area, fruit length, fruit diameter and fruit weight were lowest could be described as the best cultivars amongst others. Morphological and yield parameters of the CMV-infected plants were adversely affected by the virus and this resulted in the observed significant reductions. This is in agreement with the findings of Green (1992) who encountered severe losses in chilli plants infected with the virus. The better performance of uninoculated plants over the infected ones revealed that CMV could be devastating on the evaluated crops (okra, pepper and tomato) cultivars. Growth reduction in the viral infected plants was probably due to alterations of metabolic and physiological activities in diseased plants. This had been demonstrated in tomato plants infected with Potato virus X (PVX) and Tomato mosaic virus (ToMV) (Balogun, 2003) . The variation in fruit weight showed that as with the growth parameters, uninoculated (control) plants generally had higher biological yield values than viral infected plants. Fruit length and diameter had an obvious relationship with fruit size. The effects observed in inoculated okra, pepper and tomato cultivars could be attributed to the severity of CMV on fruit formation. In addition, the low level of growth and development of the susceptible plants agrees with Aeni (2007) who reported that the net assimilation of the virus-infected chilli plants was much lower than that of the healthy plants with substantial lower yield in diseased plants.
CONCLUSION AND RECOMMENDATIONS This study revealed that plant height, leaf area, fruit length, fruit diameter and fruit weight were all reduced by Cucumber mosaic virus in okra, pepper and tomato. The Ex-Bassawa-2 cultivar of okra, Cayenne of pepper and Roma Savanna of tomato were the most tolerant to CMV. Clemson Spineless cultivar of okra and Tropimech of tomato were moderately tolerant. The UC82B cultivar of tomato was mildly tolerant while NHAe 47-4 of okra, Yolo Wonder and California Wonder of pepper were highly susceptible to CMV. It can be concluded, therefore, that the crop cultivars responded differently to infection, indicating that further breeding efforts should be intensified. This may eventually lead to the development of completely resistant (immune) cultivars that could also be introduced to vegetable growers in the nearest future. The okra cultivar Ex-Bassawa-2, pepper cultivar Cayenne and tomato cultivar Roma Savanna could serve as sources of CMV-tolerant genes for breeding purposes. These could be crossed with susceptible genotypes for genetic improvement against the stresses imposed by CMV. Seed companies and Agricultural Development Programmes (ADPs) could multiply the seeds of these cultivars so that farmers would have easy access to highly productive seeds with disease tolerant genes. The pepper cultivar, Yolo Wonder infected with CMV suffered total crop loss. Hence, farmers should be careful to acquire seeds from reliable sources. Since CMV has been very successful in rapidly adapting to new hosts and environments, adoption of CMV resistant cultivars is very essential. Therefore, the three most tolerant cultivars (Ex-Bassawa- Table 1 : 1 Disease incidence on inoculated okra, pepper and tomato cultivars for the first two weeks after inoculation (WAI) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Disease incidence (%) Screenhouse Field Crop Cultivar 1 WAI 2 WAI 1 WAI 2 WAI Okra Clemson Spineless 283 90.8 28.3 91.3 Ex-Bassawa-2 10.0 19.2 16.7 22.0 NHAe 47-4 37.5 95.8 33.3 96.1 LSD0.05 5.7 5.4 8.2 3.5 P-value * * * * Pepper California Wonder 44.2 96.3 50.3 98.3 Cayenne 20.0 30.8 28.3 31.3 Yolo Wonder 82.5 99.2 100.0 100.0 LSD0.05 6.8 1.5 3.3 1.0 P-value * * * * Tomato Roma Savanna 0 16.7 0 18.1 Tropimech 25.0 59.2 18.3 56.7 UC82B 30.8 83.3 30.6 81.7 LSD 0.05 4.2 0.1 3.3 0.1 P-value * * * * *Significant at p=0.05
Table 2 : 2 Disease severity on inoculated okra, pepper and tomato cultivars from 2 to 6 weeks after inoculation (WAI) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Disease severity Screenhouse Field Crop Cultivar 2 WAI 4 WAI 6 WAI 2 WAI 4 WAI 6 WAI Okra Clemson Spineless 2.5 3.8 3.9 2.4 3.2 3.8 Ex-Bassawa-2 1.1 1.5 1.8 1.2 1.6 1.9 NHAe 47-4 2.9 3.7 4.1 2.8 3.6 4.0 LSD0.05 0.2 0.1 0.2 0.5 0.2 0.2 P-value * * * * * * Pepper California Wonder 3.1 3.7 4.1 3.4 3.8 4.2 Cayenne 1.7 2.7 2.9 1.5 2.2 2.6 Yolo Wonder 3.4 3.8 4.2 4.2 5.0 5.0 LSD0.05 0.1 0.1 0.2 0.1 0.1 0.1 P-value * * * * * * Tomato Roma Savanna 1.1 1.4 1.6 1.2 1.5 1.7 Tropimech 1.7 2.4 3.0 1.6 2.3 2.9 UC82B 2.2 2.9 3.4 1.8 2.5 3.1 LSD0.05 0.2 0.1 0.1 0.1 0.2 0.2 P-value * * * * * * *Significant at p=0.05
Table 3 : 3 Plant height from inoculated okra, pepper and tomato cultivars and uninoculated (healthy) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Plant height (cm) Screenhouse Field Reduction Reduction Crop Cultivar Healthy Infected LSD P-value (%) Healthy Infected LSD P-value (%) Okra Clemson Spineless 47.4 35.5 2.4 * 25.1 58.3 35.8 2.2 * 38.6 Ex-Bassawa-2 36.8 36.2 1.2 NS 1.6 40.3 39.7 0.3 * 1.5 NHAe 47-4 62.0 36.8 3.8 * 40.6 72.1 45.3 3.4 * 37.2 Pepper California Wonder 31.2 17.5 3.8 * 43.9 35.1 19.7 1.9 * 43.9 Cayenne 35.5 33.2 3.2 NS 6.5 39.9 39.6 0.1 * 0.8 Yolo Wonder 28.1 9.6 4.6 * 65.8 30.9 0 4.0 * 100.0 Tomato Roma Savanna 50.3 48.6 2.8 NS 3.4 60.1 56.0 5.7 NS 6.8 Tropimech 59.7 40.7 2.7 * 31.8 67.0 52.5 6.3 * 21.6 UC82B 54.7 30.1 1.8 * 45.0 52.1 34.6 6.2 * 33.6 *Significant at p=0.05; NS = Not Significant
Table 4 : 4 Leaf area from inoculated okra, pepper and tomato cultivars and uninoculated (healthy) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Leaf area (cm2) Screenhouse Field
Table 5 : 5 Fruit length from inoculated okra, pepper and tomato cultivars and uninoculated (healthy) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Fruit length (cm)
Table 6 : 6 Fruit diameter from inoculated okra, pepper and tomato cultivars and uninoculated (healthy) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Fruit diameter (cm) Screenhouse Field
Table 7 : 7 Fruit weight from inoculated okra, pepper, and tomato cultivars and uninoculated (healthy) with Cucumber mosaic virus under screenhouse and field conditions in Minna, Nigeria during the 2014 cropping season Fruit weight (g) Screenhouse Field
cultivar of okra, Cayenne of pepper and Roma Savanna of tomato) are recommended to farmers in CMV prone areas.
|
10.1007/s10803-019-04061-6
|
cc-by
| null | null |
openalex
|
In two studies, we investigated the effectiveness of parent education in Pivotal Response Treatment (PRT) on parent-created opportunities and spontaneous child initiations in two community-based treatment facilities for children with autism spectrum disorder (ASD). Changes in parental stress and self-efficacy were explored. Participants were 26 parents and their children who participated in group (Study 1) or individual (Study 2) parent education in PRT. Results indicated that group-based parent education resulted in moderate increases in opportunities, functional initiations, and empathic social initiations. Furthermore, parental stress reduced and self-efficacy increased. Individual parent education resulted in large increases in opportunities and functional initiations, but parental stress and self-efficacy did not change. Implications for clinical practice and directions for future research are discussed.Introduction Parenting a child with autism spectrum disorder (ASD) is more demanding than parenting a typically developing child or a child with other developmental disabilities (Hayes and Watson 2013) . ASD is a neurodevelopmental disorder characterized by impairments in social communication and social interaction, and restricted, repetitive, and stereotyped behaviors (American Psychiatric Association 2013). Many children with ASD also exhibit challenging behaviors such as tantrums, aggression, and self-injurious behavior (Jang et al. 2011; Matson et al. 2009) . Furthermore, approximately 70-92% of the children with ASD meet criteria for at least one comorbid psychiatric diagnosis, including attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), anxiety disorders, and mood disorders (Brookman-Frazee et al. 2018; Joshi et al. 2010; Simonoff et al. 2008) . The majority of children with ASD have intelligence quotient (IQ) scores above 70 and are thus not classified as having an intellectual disability (Baio et al. 2018; Joshi et al. 2014) . The characteristics of ASD and associated challenging behaviors and comorbid psychopathology not only impact children with ASD but also their parents (Karst and Van Hecke 2012) . Parents of children with ASD report more parental stress, lower levels of parental self-efficacy, and less overall well-being (e.g., Frantz et al. 2018; Hayes and Watson 2013; Karst and van Hecke 2012) . Furthermore, researchers have identified higher prevalence of depression and anxiety among parents of children with ASD (e.g., Bitsika and Sharpley 2004; Frantz et al. 2018; Singer 2006) . When the severity of ASD symptoms, challenging behaviors, or comorbid psychopathology exceed the ability of parents to cope, the likelihood of psychiatric hospitalization or inpatient treatment increases (Mandell et al. 2012; Righi et al. 2018) . Indeed, approximately 6% of children with ASD receive inpatient treatment (Cidav et al. 2013) . In early childhood, children with ASD engage in fewer initiations than typically developing children and their initiations serve fewer functions (Stone et al. 1997; Wetherby and Prutting 1984) . These deficits in initiations continue beyond early childhood. School-aged children, adolescents, and adults with ASD initiate social conversation less often which may interfere with the development of social relationships (Hauck et al. 1995; Koegel et al. 2016b; Stone and Caro-Martinez 1990) . In addition, deficits in initiations often lead to directive parent behaviors because parents tend to compensate for their child's lack of initiations rather than providing him or her with opportunities to initiate (Hudry et al. 2013; Wan et al. 2012 ). Children's challenging behaviors might also contribute to directive parent behavior and further reduce children's opportunities to initiate (Reed and Osborne 2014; Shawler and Sullivan 2017) . Parent education has long been accepted as a beneficial method to teach parents skills to improve their child's skills and reduce their child's challenging behaviors (e.g., McConachie and Diggle 2007; Steiner et al. 2012) . As a collateral result, reductions in parental stress and increases in parental self-efficacy may occur (Brookman-Frazee et al. 2009; Da Paz and Wallander 2017; Steiner et al. 2012) . Parent education also increases intervention intensity because parents are able to provide intervention throughout the day and in various natural settings, increasing the child's rate of progress and promoting generalized use of skills (Steiner et al. 2012 ). In addition, due to the increase in the number of children diagnosed with ASD, parent education is necessary to meet increased demands for treatment services (Elsabbagh et al. 2012; Steiner et al. 2012) . Parent education is an essential component of Pivotal Response Treatment (PRT; Koegel et al. 2016a) . PRT is a naturalistic evidence-based intervention that targets pivotal skills (e.g., initiations) in children with ASD to produce generalized improvements across domains of functioning using the principles of Applied Behavior Analysis (ABA). A systematic review has reported evidence for the effectiveness of PRT for increasing initiations and producing collateral improvements in communication, language, affect, play, and challenging behaviors (Verschuur et al. 2014) . Although the effectiveness of PRT has mainly been demonstrated in preschool children with ASD and cognitive impairments, some studies indicate that school-aged children with average cognitive abilities may also benefit from PRT (e.g., Doggett et al. 2013; Huskens et al. 2012; Verschuur et al. 2017) . A large number of studies on PRT focused on parent education as PRT is designed to be conducted in natural environments (e.g., Bradshaw et al. 2017; Coolican et al. 2010; Hardan et al. 2015; Nefdt et al. 2010; Randolph et al. 2011) . Parents are taught to create opportunities for their child to initiate individually, in a group, or through a self-directed learning program. Results of studies on parent education in PRT suggest that parents can be taught to implement PRT. Most parents meet criteria for fidelity of PRT implementation after parent education (e.g., Verschuur et al. 2014 ). However, in most studies, parent education in PRT was conducted by trainers who were employed by university-based research clinics. An exception is the study by Bryson et al. (2007) in which community service providers were taught to educate parents in PRT, but this study did not use an experimental design and presented only preliminary findings. To allow for dissemination of PRT to a large number of children it is important that parent education can be effectively delivered by PRT trainers who are part of communitybased treatment facilities (e.g., Brookman-Frazee et al. 2012b; Bryson et al. 2007 ) and thus more research on this topic is warranted. In addition, it is important to determine the effectiveness of parent education in PRT for parents of school-aged children with ASD, average cognitive abilities, and comorbid psychopathology, as there is limited information about the effectiveness of PRT in this population (e.g., Verschuur et al. 2017) . Furthermore, thus far there is little evidence for collateral changes in parental stress and parental self-efficacy as a result of parent education in PRT (Verschuur et al. 2014) . Finally, additional research on the effectiveness of group parent education in PRT is warranted, because only a couple of studies using a group model were conducted (e.g. Bryson et al. 2007; Gengoux et al. 2015; Hardan et al. 2015; Minjarez et al. 2011 Minjarez et al. , 2013)) . Although these studies reported improvements in parent and child behaviors, conclusive evidence for the effectiveness of groupbased parent education in PRT is still limited, as Bryson et al. (2007) and Minjarez et al. (2011 Minjarez et al. ( , 2013) ) did not use an experimental design. To address these needs, the objective of the present study was to investigate the effectiveness of parent education in PRT on parent-created opportunities and child initiations in two community-based treatment facilities for children with ASD in the Netherlands. Furthermore, collateral changes in parental stress and parental self-efficacy were explored. To this end, we conducted two separate single-case design studies of parent education in PRT. In Study 1, the effectiveness of a group parent education program was evaluated. In Study 2, we investigated the effectiveness of individual parent education. Kort et al. 2005) or the Snijders-Oomen nonverbal intelligence scale-revised 21⁄2-7 (SON-R 21⁄2-7; Tellegen et al. 1998 ). Only children with IQ scores above 70 were included because the specialized treatment center did not provide treatment to children with intellectual disabilities. Additionally, if a child received inpatient treatment during the period of data collection, the child and his or her parents were required to meet at least once every week at home or at the treatment facility. Children were not excluded if they received interventions in addition to PRT (e.g., milieu therapy, speech-language therapy, art therapy, family therapy, pharmacological interventions, or physiotherapy), as all children received concomitant interventions during their outpatient, day, or inpatient treatment at the treatment facility. However, parents, child psychologists, and child psychiatrists were requested not to make any changes in those interventions during data collection. Inclusion criteria for parents were the following: (a) no experience with PRT prior to this study, (b) access to a video camera and prepared to videotape him/herself and the child, and (c) ability to travel to the treatment facility during intervention. Informed consent was obtained from all parents and assent was obtained from children older than 12 years. The study was approved by the Ethics Committee of the Faculty of Social Sciences of the Radboud University, Nijmegen, the Netherlands (ECG2013-1304-100). Child psychologists and child psychiatrists selected 31 parents and children for participation in group-based parent education in PRT. Of those, nine parents indicated that they did not want to participate in a parent education program. Six parents stated that they were willing to participate in parent education but gave no consent for participation in the study. The remaining 16 parents agreed to participate in group-based parent education and the study. Of those, three parents did not collect sufficient baseline data and were therefore excluded. Therefore, 13 parents and their children with ASD were included in the study. Parents had a mean age of 43:8 years (SD = 5:7, range 36-52) and were primarily mothers (n = 9, 69%). Most parents were married or cohabiting (n = 11, 85%). Parent education ranged from high school (n = 7, 54%) to higher professional education or university (n = 6, 46%). Parents worked full-time (n = 4, 31%), part-time (n = 1, 8%), or were unemployed (n = 6, 46%). Two parents had another employment status (e.g., volunteer work). Children were aged between 8 and 14 years (M = 11:2, SD = 2:2) and were primarily boys (n = 10, 77%). All children had a clinical diagnosis of ASD according to the DSM-IV-TR or DSM-5 criteria. Mean score on the SRS was 82.2 (SD = 10.1, range 66-103) and average ADOS severity score was 6.8 (SD = 1.8, range 4-10). Seven children had one or two comorbid psychiatric disorders, including ADHD (n = 5), anxiety disorder (n = 1), posttraumatic stress disorder (n = 1), and ODD (n = 1). On average, children had a total IQ score of 98.0 (SD = 12.6, range 77-116), a verbal or reasoning IQ score of 99.7 (SD = 13.6, range 81-122), and a performance IQ score of 96.0 (SD = 13.3, range 75-113). Children received outpatient treatment (n = 1, 8%), day treatment (n = 5, 38%) or inpatient treatment (n = 7, 54%).
Design A multiple baseline design across four groups of participants was used to investigate the effectiveness of group parent education in PRT on parent-created opportunities and child initiations (Kazdin 2011) . Parents were randomly assigned to the first three groups and baseline started concurrently for these groups. For the fourth group, baseline started after the third intervention session of the third group. Parents were not randomly assigned to the fourth group. Participants remained in baseline for four, six, or eight weeks. Pre-tests and post-tests were conducted to explore the effectiveness of group parent education on parental stress and self-efficacy.
Procedures Baseline Baseline consisted of four to eight 10-min sessions. The purpose of baseline sessions was to assess the rate of parent-created opportunities for initiating prior to parent education in PRT and to assess the baseline level of child initiations. Baseline sessions were conducted at home and at the treatment facility if the child received inpatient treatment. During baseline, the parent did an age-appropriate everyday activity with the child that required interaction (e.g., playing a game, baking, or drawing). If the child initiated an interaction, the parent was instructed to respond as s/he usually did. Parents did not receive feedback on implementation of PRT techniques. They were asked to videotape baseline sessions using a video camera, tablet, or smartphone. Parents were instructed to record sessions (a) during which the parent, child, and activity were visible and audible on camera, (b) recorded in a one-to-one situation, and (c) lasting at least 10 min. If an activity lasted more than 10 min, parents were instructed to complete the activity. In addition, parents were asked to fill in two questionnaires during the last 2 weeks of baseline to measure parental stress and self-efficacy (see "Collateral Changes"). Intervention Group parent education was conducted by two child psychologists who worked at the specialized treatment center for children with ASD and were certified at PRT level III (i.e., demonstrated fidelity of PRT implementation at or greater than 80% with three children across three different activities). They had participated in staff training in PRT that was conducted by two PRT supervisors who were certified by the Koegel Autism Center. Each child psychologist had at least one year of experience in providing PRT. They were trained in providing parent education by the first author during a 3-h workshop. The group parent education program consisted of eight 2-h group sessions and two 60-min individual sessions (Hardan et al. 2015; Minjarez et al. 2011) . Sessions were conducted biweekly at the treatment facility. Groups were made up of both parents who did and did not consent to participate in this study. Groups consisted of seven to nine parents. From each group, two (group 3), three (group 2 and 4), or five parents (group 1) participated in this study. During group session 1-4, parents received instruction in PRT techniques (i.e., incorporating the child's choice, gaining the child's attention, providing clear opportunities, using contingent and natural reinforcement, reinforcing attempts, and interspersing maintenance and acquisitions tasks) via lectures, video-examples, worksheets, and role-plays. During the second group session, a (baseline) videotape was watched to assess the child's current level of initiations and parents were taught to set goals for their child related to initiations. PRT had originally been developed to target functional initiations (e.g., protesting and requesting objects, help, or information), but in the present study parents also received instruction in techniques to elicit social initiations (e.g., requesting social information and commenting; Doggett et al. 2013) . After each session, parents were instructed to practice PRT techniques during everyday activities with their child at home or at the treatment facility and videotape these sessions. During the first and second group session, parents did not receive feedback on their implementation of PRT in these videotapes, because the parents did not receive instruction in 'incorporating the child's choice' and 'gaining the child's attention' until the second group session. From the third group session, trainers provided feedback on parents' implementation of PRT using the following video feedback protocol: (a) trainers and parents watched approximately 1 min of a videotape, (b) trainers provided the parent with positive performance-based feedback and praise for correct use of PRT techniques, (c) for incorrect or no use of the PRT techniques, trainers provided the parent with corrective performance-based feedback, asked the parent how he or she could have correctly used PRT techniques, and provided suggestions for correct use of PRT techniques if the parent did not give a correct answer, and (d) after watching about 5 min of a videotape, trainers concluded with a positive general comment on the parent's performance (e.g., Robinson 2011 ). Individual sessions were conducted after the fourth and seventh group session. During these sessions, the child's goals were evaluated and adjusted if necessary. Parents also received feedback on their implementation of PRT. During the eighth group session, the group parent education program was evaluated and parents were asked to fill in a questionnaire to assess the social validity of the group parent education program and PRT in general.
Post-intervention We conducted three 10-min sessions immediately post parent education. Procedures were similar to those during baseline. During a post-intervention session, the parent did an age-appropriate everyday activity with the child that required interaction. Parents were instructed to implement PRT techniques but did not receive feedback on PRT implementation. The purpose of post-intervention sessions was to assess whether parents and children maintained their skills immediately after parent education. Four parents videotaped only one or two post-intervention sessions. Parents were also asked to fill out two questionnaires during the first 2 weeks of post-intervention to measure parental stress and self-efficacy (see "Collateral Changes").
Dependent Measures Parent-Created Opportunities An event-recording system was used to measure parent-created opportunities for initiating (Cooper et al. 2013) . A correct parent-created opportunity consisted of a sequence of correctly implemented PRT techniques (see Huskens et al. 2012; Verschuur et al. 2017) . Three sequences were considered correct: (1) parent presenting a clear opportunity, child initiating, and parent reinforcing the child's initiation contingently and naturally, (2) parent presenting a clear opportunity, parent prompting the child to initiate, child initiating, and parent reinforcing the child's initiation contingently and naturally, and (3) parent presenting a clear opportunity, parent prompting the child to initiate at least once, child not initiating, and parent providing no reinforcement. Operational definitions of these behaviors are presented in Online Resource 1 (Carter and Hotchkis 2002; Huskens et al. 2012; Stahmer et al. 2011b; Verschuur et al. 2017 ). An example of a correct opportunity for a functional initiation would be the parent placing the mixer on a high shelf when making cupcakes with the child and immediately giving the child the mixer when he or she requested the mixer. An example of a correct opportunity to elicit a social initiation would be the parent stating 'I'm going to cook something delicious tonight' and answering that he or she will make lasagna when the child asked 'What are we going to eat?' An opportunity would also be correct if the parent controlled the bubble blower, prompted the child to ask for bubbles by saying 'bubbles,' and did not blow bubbles if the child did no reasonable attempt to say 'bubbles'. Ten minutes of each videotape were viewed and scored by observers naïve to the phase in which videotapes were recorded (i.e., baseline, intervention, or post-intervention). If videotapes lasted more than 10 min, 10 min in the middle were observed. The entire videotape was observed if a videotape lasted less than 10 min. Nine percent of videotapes lasted less than 10 min. Observers were instructed to record each sequence using numbers (e.g., 1 = statement, 2 = verbal model prompt, 3 = child initiation, and 4 = reinforcement) and to record the time point at which the parent presented a clear opportunity to determine interobserver agreement (see Interobserver agreement). Because 9% of the videotapes lasted less than 10 min, a rate of parent-created opportunities per minute was calculated by dividing the number of opportunities by the number of minutes.
Spontaneous Child Initiations Spontaneous child initiations were measured using event-recording (Cooper et al. 2013) . A spontaneous initiation was recorded if the child began or directed a social interaction to get a response from the parent without being prompted to initiate (Carter and Hotchkis 2002; Hauck et al. 1995; Wetherby 1986 ). The child began a social interaction if his or her verbal utterance occurred at least 5 s after parent's last utterance or marked a change in conversation partner or activity. The child directed a social interaction if s/he changed or expanded the topic of conversation. Utterances that were part of an activity (e.g., 'Does she have brown eyes?' in the game Who is it?), play sounds (e.g., 'vroom vroom' when playing with cars), self-verbalizations, or echolalia were not recorded. The function of each spontaneous initiation was recorded. Three functions were distinguished: functional, early social, and empathic social (Koegel et al. 2016b; Rieth et al. 2014) . A functional initiation was recorded if the child's utterance had the purpose of regulating the parent's behavior, for example requesting objects (e.g., 'Can I have the ball?'), requesting help (e.g., 'Can you help me?'), or protesting (e.g., 'Will you stop it?'). Initiations were recorded as 'early social' if the child's utterance had the purpose of initiating social conversation and called upon the parent's involvement or interest, for example calling the parent's attention (e.g., 'Mummy, look at my drawing!') or giving information (e.g., 'My classmate invited me to his birthday party next week'). An empathic social initiation was recorded if the child's utterance had the purpose of initiating social conversation and indicated the child's interest in the parent, for example seeking social information (e.g., 'What movie did you see?'), or commenting (e.g., 'That sounds great'). Naïve observers viewed and scored 10 min of the videotapes. Observers were instructed to record the child's initiation, the function of the initiation, and the time point at which the child initiated. A rate of spontaneous child initiations per minute was calculated for each function by dividing the number of spontaneous initiations by the number of minutes.
Collateral Changes In order to explore whether groupbased parent education in PRT led to collateral changes in parental stress and self-efficacy, two questionnaires were administered during baseline and post-intervention. The Dutch version of the Parenting Stress Questionnaire (PSQ) was used to measure parental stress (Vermulst et al. 2015) . The PSQ is a 34-item questionnaire consisting of five subscales: (a) parent-child relationship problems, (b) parenting problems, (c) depressive mood, (d) parental role restriction, and (e) physical health problems. Items were rated on a four-point scale ranging from 'not true' to 'very true'. Based on subscale scores a total score was calculated. Higher total scores indicate higher levels of parental stress. Evaluation of the psychometric qualities of the PSQ showed that the construct validity and internal consistency were good (Veerman et al. 2014) . The Dutch translation of the 'Parental self-efficacy in the management of Asperger syndrome' questionnaire was used to measure parental self-efficacy (Sofronoff and Farbotko 2002) . This 15-item questionnaire describes 15 problem behaviors that are common in children with ASD (e.g., 'When your child follows routines rigidly'). Parents indicated for each behavior whether their child displayed that behavior in the previous month and rated their confidence in managing the behavior on a six-point scale ranging from 0 (no confidence) to 5 (complete confidence). An average self-efficacy score was calculated by dividing the total rating of confidence by the number of problem behaviors displayed in the previous month.
Social Validity During the last session of the parent education program, parents were asked to fill in a questionnaire to assess the social validity of group-based parent education and PRT in general. The questionnaire consisted of 29 statements (e.g., 'The trainer's feedback on my videotapes was useful' and 'The parent education program in PRT met my expectations') that were rated on a five-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). These items measured parents' attitude towards PRT, whether they judged the components of PRT parent education as informative and pleasant, and whether they considered the PRT parent education program to be effective. Finally, parents were asked to give an overall grade between 1 and 10 to rate the parent education program in PRT.
Interobserver Agreement To determine interobserver agreement, 34% of the videotapes were coded by an independent and naïve second observer, approximately evenly distributed across parent-child dyads and study phases. Prior to coding videotapes, observers were trained to use the recording systems for parent-created opportunities and child initiations. Observer training included (a) discussion of definitions, examples, and non-examples using written guidelines and video examples, (b) practice of coding using 1-min segments, (c) discussion of any discrepancies and revision of written guidelines as necessary, and (d) independent coding of 10-min training videotapes (Ledford et al. 2018) . Observer training continued until interobserver agreement was acceptable in two consecutive training videotapes (Cicchetti et al. 2006; Kennedy 2005) . Ongoing training sessions were held every 3 weeks to minimize observer drift. For parent-created opportunities, interobserver agreement was determined using mean count-per-interval (i.e., the average percentage of agreement across intervals; Cooper et al. 2013) . Videotapes were divided into 10 one-minute intervals and for each interval a percentage of agreement between counts of both observers was calculated. Mean overall percentage of agreement (i.e., across videotapes) was 89% (SD = 13; range 40-100), indicating good interobserver agreement (Kennedy 2005 ), but interobserver agreement was less than 80% for 11% of the videotapes, indicating interobserver agreement was insufficient in several instances. For spontaneous child initiations, interobserver agreement was determined using total count (Cooper et al. 2013) . Total count interobserver agreement was calculated by dividing the lower count by the higher count multiplied by 100. Mean interobserver agreement using total count was 83% (SD = 15; range 33-100), indicating good interobserver agreement. However, 29% of the videotapes had interobserver agreement below 80%, indicating that interobserver agreement was insufficient in many videotapes. Based on spontaneous initiations that were recorded by both observers, interobserver agreement on the function of these initiations was assessed per category using prevalence-adjusted and bias-adjusted kappa (PABAK; Byrt et al. 1993) . Mean PABAK was .77 (SD = .20), indicating good interobserver agreement (Cicchetti et al. 2006) .
Treatment Integrity An independent observer (i.e., a research assistant) collected data on treatment integrity based on audio recordings of the group sessions across the four groups using checklists. These checklists listed the parent education program components that were required to be implemented during a session (e.g., 'The trainers provided instruction in the PRT technique 'contingent and natural reinforcement' using a Pow-erPoint presentation'). For each component, the independent observer recorded if the trainers did or did not correctly execute that component. Treatment integrity was calculated per session by dividing the number of correctly executed components by the total number of components multiplied by 100. Mean overall percentage of treatment integrity was 95% (SD = 10; range 50-100).
Data-Analysis Analysis of data on parent-created opportunities and spontaneous child initiations involved visual and statistical analysis. Visual analysis was conducted to guide the statistical analysis (Parker and Vannest 2012 ) and consisted of systematic analysis of trend and level within and between subsequent phases for each participant, following the guidelines provided by Lane and Gast (2014) . If a phase consisted of less than three data-points, no visual analysis was conducted (Kratochwill et al. 2010) . Baseline trend was determined using the split-middle method of trend estimation. Median rates were compared to analyze changes in level between subsequent phases. Graphs that provide data on parentcreated opportunities and spontaneous child initiations are presented in Online Resource 2. Statistical analysis consisted of calculation of Tau novlap or Tau-U (Parker et al. 2011 ). Both are effect sizes for singlecase research that examine the proportion of non-overlap of data between two subsequent phases, but in addition, Tau-U also controls for undesirable positive baseline trends. If visual analysis indicated a strong positive baseline trend, Tau-U was calculated. Tau and the corresponding p value were calculated for the baseline/intervention-contrast and intervention/post-intervention-contrast for each participant using Single Case Research (SCR), a web-based calculator (Vannest et al. 2016) . Because post-intervention consisted of less than three data points for four participants, no Tau was calculated for the intervention/post-intervention contrast for 1 3 these participants (Kratochwill et al. 2010 ). Overall effect sizes (i.e., across parents or children) and confidence intervals were also calculated for both phase contrasts using SCR. Analyses were two-tailed and p value was set at .05. Using the guidelines of Vannest and Ninci (2015) , overall effect sizes were interpreted as small (<= .20), moderate (.21-.60), large (.61-.80), or very large (>= .81). Data on parental stress were analyzed for each parent using the reliability of change index (RCI; Jacobson and Truax 1991) . The RCI indicates whether changes in scores between baseline and post-intervention were statistically significantly greater than differences in scores that could have occurred due to random measurement error alone. The RCI was calculated using the following formula: where X 1 en X 2 represent the baseline and post-intervention PSQ total scores, S 1 the standard deviation of the reference sample, and r xx the test-retest reliability of the PSQ, as reported in the manual (Vermulst et al. 2015) . Analyses were two-tailed and p-value was set at .05. Consequently, an RCI < -1.96 indicated reliable negative change and a significant decrease in parental stress. An RCI > 1.96 indicated reliable positive change and a significant increase in parental stress (Jacobson and Truax 1991) . Data on parental self-efficacy were not analyzed using the RCI, because no test-retest reliability was available for the questionnaire. In addition to the RCI, data on both parental stress and self-efficacy were analyzed using Wilcoxon signed-rank tests to determine whether overall (i.e., across parents) statistically significant changes occurred as a result of parent education. In order to determine the magnitude of the changes, effect sizes were also calculated (Cohen 1992) . Following Cohen's guidelines, r was interpreted as small (.10), medium (.20), or large (.50). Spearman rank correlation coefficients were calculated to assess the association between changes in parent-created opportunities and other dependent measures (i.e., functional, early social, and empathic social initiations, parental stress, and self-efficacy) between baseline and intervention or post-intervention.
Results
Parent-Created Opportunities Median rates of parent-created opportunities during baseline, intervention, and post-interventions and values of Tau are provided in Online Resource 3. During baseline, the median rate of parent-created opportunities ranged from 0.00 to 0.10, indicating that parents hardly created opportunities RCI = X 2 -X 1 2(S 1 √ 1 -r xx ) for initiating prior to parent education in PRT. During intervention, the rate of parent-created opportunities increased significantly for six (out of 13) parents. The combined Tau across parents was .52 (90% CI .36-.67, p < .001), indicating a moderate significant effect of group parent education in PRT on parent-created opportunities. During post-intervention, no significant changes in the rate of parent created opportunities occurred, suggesting that parents maintained their skills after parent education.
Spontaneous Child Initiations During baseline, the median rate of functional initiations ranged from 0.30 to 1.50 (see Online Resource 3), indicating that most children already produced functional initiations prior to parent education in PRT. During intervention, the rate of functional initiations increased significantly for three (out of 13) children. The combined Tau across children was .37 (90% CI .23-.52, p < .001), indicating a moderate significant effect of group-based parent education in PRT on functional initiations. During post-intervention, the rate of functional initiations did not change significantly, suggesting that children maintained their intervention level of functional initiations. There were no significant increases in the rate of early social initiations during intervention or post-intervention. However, early social initiations decreased significantly for one child (out of 13 children) during intervention. For empathic social initiations, the median baseline rate ranged from 0.00 to 0.75 (see Online Resource 3), but most children hardly produced empathic social initiations during baseline. During intervention, the rate of empathic social initiations significantly increased for one (out of 13) children. The combined Tau across children was .26 (90% CI .12-.41, p = .003), indicating a moderate significant effect of group parent education in PRT on empathic social initiations. From intervention to post-intervention, the rate of empathic social initiations increased significantly for one child (out of nine children) and overall, most children maintained their intervention level of empathic social initiations during post-intervention. The top scatterplot in Fig. 1 presents the relationship between changes in median rates of parent-created opportunities from baseline to intervention and changes in median rates of functional, early social, and empathic social initiations. Changes in parent-created opportunities were not significantly associated with changes in functional initiations (r s = .06, p = .86), early social initiations (r s = -.03, p = .91), or empathic social initiations (r s = .30, p = .32).
Collateral Changes Parental stress reduced reliably and significantly in eight parents, did not change significantly in four parents, post-intervention (Mdn = 61), z = -2.45, p = 0.01, r = -0.48. Changes in parental stress were not significantly related to changes in parent-created opportunities (r s = .28, p = .36; see Fig. 1 ). There was a large significant increase in self-efficacy across parents between baseline (Mdn = 3.47) and postintervention (Mdn = 4.00), z = -2.59, p = 0.01, r = -0.53. Changes in self-efficacy were not significantly associated with changes in parent-created opportunities (r s = -.44, p = .15; see Fig. 1 ).
Social Validity Twelve (out of 13) parents completed the social validity questionnaire. Parents rated the group parent education program in PRT as highly informative (M = 4.39) and pleasant (M = 3.87). Components that were rated most informative were practicing at home (M = 4.73) and videofeedback (M = 4.58). Role-plays were rated least informative (M = 3.58). After group parent education in PRT, parents had a positive attitude towards PRT (M = 4.42) and indicated that group-based parent education benefited their child's development (M = 4.25) and their interaction with their child (M = 4.25). Overall, parents rated the group parent education program in PRT with an 8.00.
Study 2: Individual Parent Education in PRT
Method
Participants and Setting Study 2 was conducted in two community-based treatment facilities for children with ASD in the Netherlands. The first treatment facility was the same specialized treatment center for children with ASD as in Study 1 (i.e., Dr. Leo Kannerhuis). The second treatment facility was Youké, which is a center for youth care that also provided day treatment to children with ASD. Child psychologists and child psychiatrists in both treatment facilities referred potential participants to the study. Inclusion and exclusion criteria for parents and their children were identical to those in Study 1. The study was approved by the Ethics Committee of the Faculty of Social Sciences of the Radboud University, Nijmegen, the Netherlands (ECG2013-1304-100). Child psychologists and child psychiatrists selected 20 parents and children for participation in individual parent education in PRT. Of those, 16 parents agreed to participate in individual parent education and the study. One of these parents was excluded because the child's IQ was too low. Two parents were excluded because they did not collect sufficient baseline data. Therefore, 13 parents and their Fig. 1 Relationship between changes from baseline to intervention/ post-intervention in parent-created opportunities and functional initiations (circles), early social initiations (squares), and empathic social initiations (triangles; top), parental stress (crosses; middle), and parental self-efficacy (diamonds; bottom) for group parent education and increased significantly in one parent (out of 13 parents). Across parents, there was a medium to large significant decrease in stress from baseline (Mdn = 75) to children with ASD were included in the study to investigate the effectiveness of individual parent education in PRT. Six children received treatment at the first treatment facility and seven children were treated at the second treatment facility. Parents had a mean age of 40:6 years (SD = 7:8, range 30-55) and were primarily mothers (n = 9, 69%). Most parents were married or cohabiting (n = 10, 77%). Parent education ranged from high school (n = 6, 46%) to higher professional education or university (n = 7, 54%). Parents worked full-time (n = 3, 23%), part-time (n = 8, 62%), or were unemployed (n = 2, 15%). Children had a mean age of 6:11 years (SD = 3:2; range 3-11) and were primarily boys (n = 11, 85%). All children had a clinical diagnosis of ASD according to the DSM-IV-TR or DSM-5 criteria. Mean score on the SRS was 84.0 (SD = 8.5, range 76-109) and average ADOS severity score was 6.1 (SD = 1.9, range 3-9). Three children had one or two comorbid psychiatric disorders, including ADHD (n = 1), anxiety disorder (n = 1), ODD (n = 1), and unspecified conduct disorder (n = 1). On average, children had a total IQ score of 95.4 (SD = 14.0, range 72-119), a verbal or reasoning IQ score of 98.3 (SD = 9.6, range 87-115), and a performance IQ score of 90.2 (SD = 17.6, range 59-122). Children received outpatient treatment (n = 1, 8%), day treatment (n = 9, 69%) or inpatient treatment (n = 3, 23%).
Design Two concurrent multiple baseline designs across participants (i.e., one for each treatment facility) were used to investigate the effectiveness of individual parent education (Kazdin 2011) . Participants were randomly assigned to a baseline length and remained in baseline for four, six, or eight weeks. Pre-tests and post-tests were conducted to explore the effectiveness of individual parent education on parental stress and self-efficacy.
Procedures Baseline Baseline consisted of four to eight 10-min sessions. Procedures for baseline sessions were identical to those in Study 1. Intervention Individual parent education was conducted by a child psychologist, social worker, or direct-care staff member who worked at the first or second community-based treatment facility. Each trainer was certified at PRT level III and had at least 1 year of experience in providing PRT. Trainers were trained in providing individual parent education by the first author during a 3-h workshop. Individual parent education consisted of four 90-min sessions and six 60-min sessions. Sessions were conducted weekly at one of the treatment facilities or at the parents' home. Sessions were attended by one parent or both parents. Parents received instruction in PRT techniques via lectures, video-examples, worksheets, and role-plays during session 1-4. Children did not attend these sessions, since these sessions were designed to provide parents with instruction in PRT techniques. During the second session, a (baseline) videotape was watched to assess the child's current level of initiations and parents were taught to set goals for their child related to initiations. After each session, parents were instructed to practice PRT techniques during everyday activities with their child and to videotape these sessions. During the first and second session, parents did not receive feedback on their implementation of PRT in these videotapes, because parents did not receive instruction in 'incorporating the child's choice' and 'gaining the child's attention' until the second session. As of the third session, parents received feedback on their implementation of PRT techniques using the same video feedback protocol as was used in Study 1. Sessions 5-10 were also attended by the child. During the first 10 min of these sessions, parents practiced PRT techniques with their child during an everyday activity, which was videotaped by the trainer. These PRT sessions were conducted in case the parent had not been able to videotape a PRT session at home. During the next 30 min, parents received feedback on their implementation of PRT in the videotape recorded by the trainer and/or the videotape recorded at home. For the remainder of the session, parents practiced PRT techniques with their child during an everyday activity while receiving feedback from the trainer (i.e., guided practice) using the following protocol: (a) if the parent implemented PRT correctly, the trainer provided positive performance-based feedback and praise every minute, (b) if the parent implemented PRT incorrectly or did not implement PRT, the trainer provided corrective performance-based feedback and modeled correct use of PRT, and (c) after 20 min, the trainer concluded with a positive general comment on the parent's performance (e.g. Randolph et al. 2011) . During the fifth and ninth session, the child's goals were evaluated and adjusted. During the tenth session, the individual parent education program in PRT was evaluated and parents were asked to complete a questionnaire to assess the social validity of individual parent education and PRT in general.
Post-intervention Post-intervention consisted of three 10-min sessions that were conducted immediately post parent education. Procedures for post-intervention sessions 1 3 were identical to those in Study 1. One parent videotaped only two post-intervention sessions and another parent was not able to record any post-intervention sessions.
Dependent Measures Dependent measures were identical to those in Study 1.
Interobserver Agreement To determine interobserver agreement, 36% of the videotapes were coded by an independent and naïve second observer, approximately evenly distributed across parent-child dyads and study phases. Prior to coding videotapes, observers were trained to use the recording systems for parent-created opportunities and child initiations. This observer training was the same as in Study 1. For parent-created opportunities, interobserver agreement was determined using mean count-per-interval (Cooper et al. 2013) . Mean overall percentage of agreement was 86% (SD = 13; range 40-100), indicating good interobserver agreement (Kennedy 2005 ), but interobserver agreement was less than 80% for 28% of the videotapes, indicating that interobserver agreement was insufficient in many videotapes. For spontaneous child initiations, interobserver agreement was determined using total count (Cooper et al. 2013) . Mean interobserver agreement using total count was 79% (SD = 18; range 17-100), indicating acceptable interobserver agreement. However, 40% percent of the videotapes had interobserver agreement below 80%, indicating that interobserver agreement was insufficient in many instances. Based on initiations that were recorded by both observers, interobserver agreement on the function of these initiations was assessed per category using PABAK (Byrt et al.1993) . Mean PABAK was .77 (SD = .20), indicating good interobserver agreement (Cicchetti et al. 2006) .
Treatment Integrity Each trainer collected data on treatment integrity after each session using checklists. These checklists listed the individual parent education program components that were required to be implemented during a session (e.g., 'During videofeedback, the trainer gave positive performance-based feedback and praised the parent every minute, if he or she created one or more correct opportunities'). For each component, the trainer recorded if he or she did or did not correctly execute that component. Mean overall percentage of treatment integrity was 95% (SD = 8; range 63-100). For 21% of the sessions an independent observer (i.e., research assistant) collected data on treatment integrity based on audio recordings of the sessions to assess interobserver agreement in treatment integrity using PABAK. Mean value of PABAK was .82 (SD = .23), indicating excellent interobserver agreement on treatment integrity (Cicchetti et al. 2006) .
Data-Analysis Analyses were identical to those in Study 1.
Results
Parent-Created Opportunities The baseline median rate of parent-created opportunities ranged from 0.00 to 0.35 (see Online Resource 3), indicating that most parents did not create opportunities for initiating prior to individual parent education in PRT. During intervention, the rate of parent-created opportunities increased significantly for 11 (out of 13) parents. The combined Tau across parents was 0.80 (90% CI 0.66-0.94, p < .001), indicating a large effect of individual parent education in PRT on parent-created opportunities. During post-intervention, there was a significant increase in the rate of parent-created opportunities for one (out of 11) parents, but across parents the rate of parent-created opportunities hardly changed (Tau combined = 0.04, 90% CI -0.15-0.24, p = .71). This suggests that, overall, parents maintained their skills after parent education.
Spontaneous Child Initiations During baseline, the median rate of functional initiations ranged from 0.00 to 1.30 (see Online Resource 3), indicating that most children already produced functional initiations prior to parent education. During intervention, the rate of functional initiations increased significantly for 10 (out of 13) children. The combined Tau across children was .65 (90% CI .52-.79, p < .001), indicating a large significant effect of individual parent education on functional initiations. During post-intervention, there were no significant changes in the rate of functional initiations (see Online Resource 3), suggesting that, overall, children maintained their intervention level of functional initiations. There were no significant increases in the rate of early social initiations during intervention. However, early social initiations decreased significantly for one child (out of 13 children). During post-intervention, there was an increase in the rate of early social initiations for two (out of 11) children. For empathic social initiations, the median baseline rate ranged from 0.00 to 0.15 (see Online Resource 3), indicating that children hardly produced empathic social initiations during baseline. During intervention, the rate of empathic social initiations significantly increased for two (out of 13) children. The combined Tau across children was .13 (90% CI .00-.27, p = .10), indicating a small non-significant effect 1 3 of individual parent education on empathic social initiations. From intervention to post-intervention, there were no significant changes in the rate of empathic social initiations, suggesting that, overall, children maintained their intervention level of empathic social initiations during post-intervention. The top scatterplot in Fig. 2 presents the relationship between changes in median rates of parent-created opportunities from baseline to intervention and functional, early social, and empathic social initiations for individual parent education. There was no significant association between changes in parent-created opportunities and changes in functional initiations (r s = .27 p = .38), early social initiations (r s = .16, p = .61), or empathic social initiations (r s = .23, p = .44).
Collateral Changes Parental stress reduced reliably and significantly in three parents, did not change significantly in eight parents, and increased significantly in two parents (out of 13 parents). Across parents, there was no significant change in stress between baseline (Mdn = 61) and post-intervention (Mdn = 62), z = -0.63, p = 0.53, r = -0.12. Changes in parental stress were not significantly related to changes in parent-created opportunities (r s = .14, p = .64; see Fig. 2 ). There was no significant increase in self-efficacy across parents from baseline (Mdn = 3.73) to post-intervention (Mdn = 3.78), z = -0.82, p = 0.41, r = -0.16. Changes in self-efficacy were not significantly associated with changes in parent-created opportunities (r s = .24, p = .40; see Fig. 2 ).
Social Validity All parents completed the social validity questionnaire after parent education. Individual parent education was rated as highly informative (M = 4.42) and pleasant (M = 4.23) . With regard to the components of the individual parent education program, video-feedback and guided practice were rated most informative, with mean scores of 4.69 and 4.54, respectively. Role-plays were rated least informative, but nonetheless highly informative (M = 4.23). Parents' attitude towards PRT was positive post parent education (M = 4.23) and parents indicated that individual parent education in PRT benefited their child's development (M = 4.08) and their interaction with their child (M = 4.08). Overall, parents rated the individual parent education program in PRT with an 8.08.
Discussion In two single-case design studies, parents of children with ASD were taught to create opportunities for initiations through group or individual parent education in PRT. Collateral changes in parental stress and self-efficacy as a result of group or individual parent education were also explored. The results from the first study indicate that group-based parent education in PRT had a moderate significant effect on parent-created opportunities, functional initiations, and empathic initiations. Furthermore, parental stress significantly decreased and self-efficacy significantly increased. Finally, parents were highly satisfied with the group parent education program. The results from the second study show that parents were also highly satisfied with individual parent education in PRT. Moreover, individual parent education resulted in large significant increases in parent-created opportunities and functional initiations, but changes in parental stress and self-efficacy were not significant. In both studies, changes in parent-created opportunities were not significantly associated with changes in any other dependent measure. The results from the first study partially support the notion that parents can effectively be taught PRT in a group in a community-based treatment facility. Less than half of the parents created significantly more opportunities as a result of group-based parent education and functional and empathic social initiations increased significantly in only a few children, despite moderate effects across parents and children. One factor that could explain why our group parent education program in PRT seems less effective than those in the studies of Hardan et al. (2015) and Minjarez et al. (2011) is the age of the included children. Children of parents in our group parent education program were older (i.e., school-aged). PRT might be more difficult to implement in school-aged children, because these children play or work more independently compared to preschoolers, which might make it more difficult to create opportunities for functional initiations (Suhrheinrich et al. 2016) . Also, more than half of the children in our group parent education program had at least one comorbid psychiatric diagnosis, whereas children with comorbid psychopathology were excluded from participation in the study of Hardan et al. (2015) . Comorbid psychopathology may negatively affect the effectiveness of an intervention (e.g., Antshel et al. 2011) . Furthermore, comorbid psychopathology is often the primary reason for referral to treatment services and thus, parents may prioritize intervention targets related to these co-occurring psychiatric problems over initiations (e.g., Brookman-Frazee et al. 2012a) . Another factor that might explain the mixed results of our study on group parent education is the amount of practice and individual feedback. On average, parents who participated in group-based parent education videotaped only seven sessions during parent education, although they were instructed to videotape one PRT session after each session. Thus, parents in our group parent education program practiced less than instructed, suggesting low treatment adherence. As a result, they received less feedback. Both practice and individual feedback are critical to enhance parents' skills (e.g., Parsons et al. 2012) . It is possible that parents in group-based parent education felt little pressure to practice because they could 'hide behind' the group (Wymbs et al. 2017) . For future research it is important to determine the optimum amount and type of practice and feedback to teach parents to create opportunities for initiating and to identify strategies to increase treatment adherence in group parent education programs. Findings from our second study are consistent with previous studies on individual parent education in PRT that have shown increases in parents' ability to implement PRT and improvements in child functional verbal communication, including initiations (e.g., Bradshaw et al. 2017; Coolican et al. 2010; Koegel et al. 2002; Randolph et al. 2011; Symon 2005) . The current study provides additional evidence for the effectiveness of individualized parent education programs in PRT by demonstrating that individual parent education can be effectively implemented by trainers in community-based treatment facilities. In both studies changes in parent-created opportunities were not significantly related to collateral changes in parental stress and self-efficacy. Rather, particularly in our study on parent education using a group model, improvements in parental stress and self-efficacy seemed to occur irrespective of increases in parent-created opportunities. This suggests that it may be important to provide parents with opportunities to meet other parents of children with ASD who have similar experiences, prior or in addition to teaching skills to these parents to improve their child's skills. As such, these results support the hypothesis that participating in parent education in a group context may decrease parental stress and increase their self-efficacy, because a parent education group would increase social support (e.g., Frantz et al. 2018) . Our studies on group and individual parent education in PRT are unique in distinguishing between different subtypes of child initiations based on their communicative function compared to other studies evaluating the effectiveness of PRT on child initiations. Until now, studies on PRT targeted only one type of initiations (e.g., Koegel et al. 2010) or initiations in general without distinguishing between communicative functions (e.g., Hardan et al. 2015; Minjarez et al. 2011) . A distinction between initiations based on their communicative function is important as social initiations have more potential to improve children's social success than functional initiations (e.g., Koegel 2000) . PRT particularly provides strategies to elicit functional child initiations (e.g., shared control, waiting, and interrupting a routine), although leading statements can be used to create opportunities for empathic social initiations (e.g., Doggett et al. 2013 ). Therefore, it is not surprising that we particularly found significant increases in functional and to a lesser degree in empathic social initiations as a result of parent education in PRT. Our baseline data also suggest that children with ASD may show deficits in one subtype of initiations, but not in another subtype. These data support the notion that PRT needs to be individualized based on child characteristics and that parents need to be taught to create opportunities to target a certain subtype of initiations (Rieth et al. 2014 ). Further research is necessary to validate our distinction in subtypes of initiations. There was a great deal of variability in responding between parents and children in both studies. Parent characteristics may influence parents' intervention outcomes, including parent fidelity of intervention implementation (e.g., Randolph et al. 2011) . For example, parent education may be less effective in economically disadvantaged families, although these parents appeared to benefit significantly more from individual than group parent education models (Lundahl et al. 2006 ). Parent's cultural background, educational level, marital status, parental stress, psychopathology, and gender are also likely to be related to parent fidelity of intervention implementation (e.g., Osborne et al. 2008; Reyno and McGrath 2006; Stahmer et al. 2011a; Strauss et al. 2012 ). Further research is necessary to examine how these factors affect the effectiveness of parent education in PRT. This will enable clinicians to tailor parent education in PRT to each parent's needs and to optimize individual outcomes. Variability in responding between children may be a result of variation in parents' implementation of PRT as fidelity of implementation is associated with intervention outcomes (e.g., Allen and Warzak 2000; Strauss et al. 2012) . Furthermore, child characteristics might account for this variability. Research has indicated that higher preintervention cognitive and expressive language skills, more positive affect, more appropriate toy contact, and decreased social avoidance and stereotyped or repetitive vocalizations predict positive outcome of PRT in preschool children with ASD (Fossum et al. 2018; Schreibman et al. 2009; Sherer and Schreibman 2005) . Future research should investigate whether these and other child characteristics, such as comorbid psychopathology and challenging behavior, are related to outcomes of PRT for school-aged children with ASD. There are several limitations to both studies. First, due to limited availability of certified PRT trainers, the studies on group and individual parent education could not be conducted concurrently. As a result, parents and children were not randomly assigned to the study on group or individual parent education or matched. We conducted two separate single-case design studies and thus, differences in the effectiveness of both parent education models could not be evaluated. This topic should be addressed in future research. Second, children included in both studies were also receiving other interventions at the treatment facilities (e.g., milieu therapy, speech-language therapy, art therapy, family therapy, pharmacological interventions, or physiotherapy). For ethical reasons, it was necessary to continue these interventions and thus the possibility of multiple intervention interference cannot be ruled out. Third, although interobserver agreement was acceptable or good on average, there were several instances where interobserver agreement was below acceptable levels, which impacts the accuracy of our data. Interobserver agreement for parent-created opportunities may have been low in several instances, because a parent-created opportunity was defined as a sequence of multiple behaviors (Cooper et al. 2013 ). Because we measured spontaneous initiations in children with very different levels of verbal communication (ranging from a few words to verbally fluent) and measured different types of initiations (i.e., functional, early social, and empathic social), our definition of an initiation was relatively 'broad', which may have resulted in low levels of interobserver agreement in many videotapes (Cooper et al. 2013 ). In addition, interobserver agreement may have been low, because the audio quality of several videotapes was poor due to background noises or bad acoustics. Fourth, to practice the PRT techniques parents were allowed to choose any age-appropriate everyday activity that required interaction. This enabled parents to select child-preferred activities and to follow their child's motivation, as is expected during PRT (Koegel et al. 2016a ), but also resulted in variation in activities, which could have affected our results. Finally, collateral effects were measured only before and after the intervention using questionnaires completed by parents. Repeated measures using a combination of direct or objective assessment methods (e.g., observation or physiological measures) and indirect or subjective measures (e.g., questionnaires) would have been more suitable to measure changes in parental stress and self-efficacy (Cooper et al. 2013) . Despite the above stated limitations, this study provides support for the use of individual parent education in PRT in community-based treatment facilities to teach parents to create opportunities for initiations. Our findings also suggest that delivering parent education in a group format is moderately effective for this purpose. Also, providing parents with opportunities to meet with other parents in similar circumstances may result in reductions in parental stress and increases in self-efficacy. As current demand for treatment services for children with ASD exceeds the availability of such services and effective and efficient interventions are essential, clinicians in community-based treatment facilities may choose to combine individual parent education in PRT with group sessions. Further research is warranted to identify parent and child characteristics that affect effectiveness of parent education in PRT in order to be able to tailor interventions to meet each individual's needs and to optimize individual outcomes. conducted at the Dr. Leo Kannerhuis, which is a specialized treatment center for children with ASD in the Netherlands. Children received outpatient treatment, day treatment, or inpatient treatment because of severe autism symptoms, comorbid psychopathology, challenging behaviors, and/or difficulties in family functioning. Child psychologists and child psychiatrists referred potential participants to the study. Children were included if they met the following inclusion criteria: (a) clinical diagnosis of ASD according to the DSM-IV-TR or DSM-5 criteria (American Psychiatric Association 2000, 2013) and confirmed by scores on the Social Responsiveness Scale (SRS-2; Constantino and Gruber 2012; Dutch version by Roeyers et al. 2015) and/ or Autism Diagnostic Observation Schedule (ADOS; Lord et al. 2012; Dutch version by De Bildt et al. 2013), (b) aged between 3 and 15 years at baseline, and (c) total IQ, verbal/ reasoning IQ, or performance IQ above 70 on the Dutch version of the Wechsler Intelligence Scale for Children-III (WISC-III-NL;
Fig. 2 2 Fig.2Relationship between changes from baseline to intervention/ post-intervention in parent-created opportunities and functional initiations (circles), early social initiations (squares), and empathic social initiations (triangles; top), parental stress (crosses; middle), and parental self-efficacy (diamonds; bottom) for individual parent education
Seyscentra, De Horst 6, 6581 TE Malden, The Netherlands
|
10.20884/1.jm.2023.18.1.6833
|
cc-by
| null | null |
openalex
|
There are two species of plant that grow flourishing all around Indonesia including in Banten and West Java which are Merremia vitifolia and Bidens pilosa. In this study we evaluate how these plants could be potentially used as natural preserver of fish product especially Auxis thazard, to inhibit the histamine formation, and to find out how this activity correlates to the substances in polar extract of B. pilosa flowers and M. vitifolia leaves. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) were used to identify the substances of the plant's part extracts. There are 27 chemicals in M. vitifolia extract and 14 chemicals in B. pilosa extract that have been detected. A triglyceride has been detected, isolated, and characterized by FTIR, 1 H-NMR and 13 C-NMR from n-hexane extract of M. vitifolia supported by LC-MS/MS data. Histamine formation in fish was determined after 30 min treatment with 4hydroxybenzoic acid solution is around 40 -51 mg / 100 g of fish, while treatment with M. vitifolia and Bidens pilosa extracts were less than 10 mg / 100 g of fish. This is the indication of high potential of both extract as preserver of fish products. Many of the identified substances have bioactivity like antimicrobial, anticancer, anti-inflammatory, antioxidant, and more, which influence the extracts' ability to inhibit the formation of histamine in fish.INTRODUCTION Indonesia is an archipelago country and the highest fisheries producent country in South-east Asia. Indonesian consumed fish 47.34 kg/capita in 2017 and increasing every year (Firmansyah et al., 2019) . Pelagic type of fish is widely obtained commercially in market at many cities especially in East Indonesia and it has high economic value for export trade including Frigate Tuna (Auxis thazard). Frigate Tuna is from the family of Scombridae that have red flesh. The damage of the fish by bacterial activity as well as enzymes can produce toxin called Scombrotoxin. This toxin is histamine produced by fish because of improper refrigerated or preserved (Hattu et al., 2015) . Scombrotoxin poisoning is usually associated with consumption of fish containing high levels of histamine (Zare et al., 2015) . Histidine decarboxylase breaks down histidine into histamine in the skin, gut, and gills of the fish. There are wide range of bacteria that produce histidine decarboxylase including Pseudomonas, Citrobacter, Clostridium, Klebsiella and Morganella morganii (Pawul-Gruba & Osek, 2021) . After histamine formed in the meat of fish, it will remain active even after cooking, because histamine is heat stable. This is why, the formation of histamine should be prevented or inhibited to preserve the quality of fish. Histamine involved in human physiological functions such as local immune and inflammation responses, and neuromodulation if the body consumed high amount of histamine, it could pop up symptoms similar of allergic reaction. The typical observed symptoms are itching, nausea, vomiting, abdominal cramps, headaches, flushing and hypotension (Biji et al., 2016; Visciano et al., 2014) . Meanwhile, Indonesia is tropical country with redundant of medical plants and can be used as inhibitor of histamine production in fresh fish or any other biological activities. There are some reports on the plant's extract that possess antihistamine activity such as Tamarindus indica L. (Hattu et al., 2015) , Solanum nigrum and Ricinus communis (Lomash et al., 2010) , Endophytic Fungi in Curcuma longa L. (Lomash et al., 2010) and E. prostrata (Linn.) (Risfa et al., 2021) . There are two species of plant that grow flourishing all around Indonesia including in Banten and West Java which are Merremia vitifolia and Bidens pilosa. Both plants are known traditionally to treat several illnesses like headache, rheumatism, eye inflammation, dysentery, swollen glands, ear infections, and mouth ulcers (Akter et al., 2021; Xuan & Khanh, 2016) . The first plant is M. vitifolia which locally know in Indonesia as Bijalang Bulu or Akar Buluh, in recent years has been studied. It has immense potential as antioxidant, anti-arthritic and anti-nociceptive (Akter et al., 2021) , as inhibitor of α-glucosidase (Tahya & Karnelasatri, 2021) , and antibacterial (Hasanah et al., 2020) . But very few reports about the chemical's composition of the extracts. Previously, we have studied the chemicals composition of n-hexane extract of M. vitifolia using GC-MS (Tahya & Karnelasatri, 2021) , but in current research we study more on the chemicals composition of M. vitifolia extracts using LC-MS/MS and GC-MS. The study of chemicals compositions will open broader potential applications of this plant. In contrast to M. vitifolia, B. pilosa is a plant that has been well studied. It is originally from South America but widely distributed in most tropical and subtropical areas of the world including Indonesia. It has many names all over the world, but in Indonesia, it is known as Ketul especially in Java. The major chemicals identified in B. pilosa are polyacetylenes, triterpenes, some essential oils, and flavonoids. These chemicals are considered as the most active substances that responsible for biological and pharmacological actions of the plant. But there are very few reports in chemicals constituents of B. pilosa grow in Indonesia, especially in west part of Java Island (Silalahi et al., 2021) . In this study we evaluate how these plants could be potentially used as natural preserver of fish product especially Auxis thazard by inhibit the histamine formation. We try to find out how this activity correlates to the substances in polar extract of B. pilosa's flowers and M. vitifolia leaves that harvested in Tangerang regency, Banten province, Indonesia. To Identify the substances of the plants extract, we used liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).
EXPERIMENTAL SECTION Materials and Instruments Fresh leaves of Merremia vitifolia and all part of the flowers of Bidens pilosa were collected in Panongan District, Tangerang Regency, Banten, Indonesia. For extraction process the solvents n-hexane (Merck), ethyl acetate (Merck), and methanol (Merck) were purchased from PT. Pasifik Kimia Indonesia. Chemicals used for histamine forming inhibition test are sulfanilic acid (Merck), sodium nitrite (Merck), NaCl (Merck), sodium sulfate anhydrate (Merck), sodium phosphate monohydrate (Merck), 1-butanol (Merck), Na2CO3 (Merck), hydrochloric acid (Merck), histamine dihydrochloride (Merck), 4-hydroxybenzoic acid (Merck), TLC 60 aluminum plat F254 (Merck), distilled water, and filter paper. Analytical Balance (Adventure Pro AV264C), Electric Heater (Cimarec 2), UV lamp, Centrifuge (Labofuge 200-Heraeus), UV-Vis Spectrophotometer (Apel PD-303S spectrophotometer), GC-MS instrument (Agilent GC 6890N 5975B MSD system), LC-MS/MS instrument (Waters, Acquity UPLC I-Class with Xevo G2-XF QTof), Agilent 500 MHz NMR spectrometer with DD2 console system and FTIR Alpha Bruker.
Samples Preparation and Extraction Total of 500 g of leaves (M. vitifolia) sample and 500 g of flowers (B. Pilosa) sample were collected. Both samples were cleansed with distilled water, let it dry for 2 days at dark and room temperature. Chop each the samples into small parts and dried in oven at 80 o C for 36 hours. Samples were cut into smaller pieces with blender. As many as 80.48 g of dried rough powder of M. vitifolia leaf and 105.01 g of B. pilosa flower were obtained. Weight 20.0 g of the sample powder and added into 150 mL of solvent in 250 mL Erlenmeyer flask and tightly sealed. For M. vitifolia, sample was extracted with n-hexane and methanol, while B. pilosa sample was extracted with methanol only. Extraction took place for 24 hours in room temperature and dark condition. However, for flask containing extract of n-hexane of M. vitifolia, after 3 hours maceration was stop for string and let the mixture of liquid and solid be separated in 15 minutes. Took about 15 mL liquid extract of n-hexane of M. vitifolia and put into vial bottle and let solvent evaporate, and sample 1 ready to send for the first LC-MS/MS analysis. The flask containing the remain nhexane extract was sealed again and continue maceration for 21 hours more. Flasks containing methanol extracts were continuing maceration process for 24 hours nonstop. After maceration, most solvents were evaporated using rotary evaporator. Samples were divided into several vial bottles. The samples contain methanol extract of M. vitifolia and B. pilosa, were designated as sample 2 and 3, respectively (still containing small quantity of solvent) and send to analytical laboratory of ITB Bandung for GC-MS analysis. Other samples also contained methanol extract of M. vitifolia and B. pilosa were designated as sample 4 and 5 respectively, were sent to LC-MS/MS analysis. Sample 6 contained n-hexane extract of M. vitifolia was divided into 2 vial bottles (6a and 6b) and let to dry (until free solvent) for 2 more days in dark place at room temperature. One dried sample (6a) was sent to LC-MS/MS analysis at Center of Chemistry Research, Indonesian Institute of Sciences, and the other dried sample (6b) was prepared for TLC preparative.
Isolation of Triglyceride A dry and clean TLC 60 aluminum plat F254 size 20 x 20 cm 1 mm thick with based line was prepared. About 3 mL n-hexane solvent to dry extract in vial bottle and mix it. Pipetted all the mixture of extract into the base line of TLC plat and let for air dry. Put TLC plat into chamber contain solvents mixture of nhexane: ethyl acetate (8:1). After separation of substance in TLC plat, the yellow substance was scraped from the silica layer of TLC. The yellow power was dissolved in n-hexane 10 mL, and then filtered with Whatman 40. The yellow solution was dried for several hours until 1/3 volume remain. The solution was then spotted onto new, dry, and clean TLC 60 aluminum plat F254 size 5 x 10 cm at the base line, then run in the chamber with similar solvent. Any spots were observed under UV light. The isolated fraction was analysis with NMR and FTIR for structure identification.
Preparation of 4-Diazoniobenzenesulfonate Reagent A mixture of 1.5 mL of 0.9% (w/v) sulfanilic acid in 4% HCl and 1.5 mL of 5% (w/v) NaNO2 was immersed in ice water for 5 minutes. 6 mL of 5% NaNO2 solution was added and allowed to stand for 5 minutes. The volume was adjusted to 50 mL with cold distilled water. Then, the reagents were kept in an ice bath for 15 minutes. Then let stand for 12 hours and ready to use (Hattu et al., 2015) .
Histamine Standard Curve Histamine dihydrochloride was dissolved in 100 mL of distilled water for concentration of 1000 ppm. The standard histamine solution of 1000 ppm was then diluted with distilled water to obtain a concentration of 100 ppm, which was then diluted to obtain concentrations of 5, 20, 40, 60, 80 and 100 ppm. A total of 1 mL of histamine standard solution. Every standard solution with concentrations of 5, 20, 40, 60, 80 and 100 ppm, was reacted with 4diazoniobenzenesulfonate and the absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 496 nm. A curve of absorbance versus histamine concentration was made (Hattu et al., 2015) .
Pre-treatment of Fish with Extracts Powders of M. vitifolia and B. pilosa, and 4hydroxybenzoic acid were weighed 5 g for 5% concentration, 10 g for 10% concentration, and 15 g for 15% concentration for each one of them, and then mixed into 100 mL distilled water at constant temperature of 60 o C and extracted for 1 hours. Then filtered with filter paper. The Frigate Tuna fish are washed, the dorsal part of the fish (without the skin) is taken from the fish body. Then it was sliced into small pieces, took 10 g of it, and soaked in aqueous extract of M. vitifolia, B. pilosa, and solution of 4hydroxybenzoic acid at various concentrations for 30 minutes and then washed it with distilled water.
Histamine Extraction Thin slices of Frigate Tuna fish meat weighed as many as 5.0 g. The sample was homogenized with 20 mL of 0.85% (w/v) NaCl solution for 2 minutes using a blender. Then it was put into a 75 mL centrifuge tube and centrifuged at 5300 rpm for 1 hour. The supernatant formed was made into 25 mL with 0.85% NaCl solution. The extract was used for further analysis. In a test tube, 1 mL of the extract was diluted to 2 mL with a solution of 0.85% NaCl and 0.5 g of a salt mixture (containing 6.25 g of anhydrous Na 2 SO 4 to which 1 g of Na3PO4.H2O), the solution was mixed evenly. Then 2 mL of n-butanol was added and shaken vigorously for 1 minute and allowed to stand for 2 minutes and homogenized so that the protein gel was damaged. The tube was then shaken for a few minutes and centrifuged at 3100 rpm for 10 minutes. Butanol located at the top (about 1 mL) was transferred into a clean and dry tube. Then evaporated to be completely dry. The residue was dissolved in 1 mL of distilled water and ready for next step (Hattu et al., 2015) .
Histamine Content Analysis A clean test tube containing 5 mL of 1.0% Na2CO3 solution was slowly added to 2 mL of 4diazoniobenzenesulfonate reagent and mixed. Then added 1 mL of the residual solution obtained from the extraction process into the tube. The absorbance of the resulting color was measured immediately after 5 minutes at a wavelength of 496 nm using distilled water as a blank (Hattu et al., 2015) . The increasing of histamine content was calculated by equation 1.
RESULTS AND DISCUSSIONS Chemicals Identification by LC-MS/MS in n-Hexane Extract of M. vitifolia The LC-MS/MS analysis of n-hexane extract of M. vitifolia has been conducted. The chromatogram results for sample 1 and 6a are shown in Figure 1 and Figure 3 . Table 1 dan 2 shows all the proposed compound that identified by LC-MS/MS analysis in nhexane extract of M. vitifolia which are from sample 1 and 6a, respectively. From LC-MS/MS analysis, there are 12 compounds that has been identified in sample 1 and there are 4 compounds identified in sample 6a. The identification using Internal Library of the instrument (Waters), or based on the literature research, and based on the mass spectrum or exact mass of the compounds with higher similarity to the database of natural chemical products in NCBI PubChem. Total, we identified and proposed 16 chemical's names from n-hexane extract of M. vitifolia. Figure 2 . One orange-yellowish spot was observed under UV light on TLC plat after the TLC preparative procedure was conducted. 2 . This isolated fraction 1 was analyzed with FTIR, 1 H-NMR dan 13 C-NMR to identify most possible compound. FTIR of the isolate fraction was shown in Figure 4 . The FTIR spectral data shows vibration bands at 3011.07 cm -1 (=C-H olefinic), 2923.69 cm -1 (-CH2-sym stretch), 2851.67 cm -1 (-CH2-asym stretch), 1742.42 cm -1 (-C=O stretch), 1461.97 cm -1 (-CH2 deform), 1377.93 cm -1 (-CH3 deform), 1236.18 cm -1 (-O-C=O), and 1168.24 cm -1 (-CH-O stretch). Many of these vibration bands have similarity to the wave number of FTIR spectral of triglycerides (Harry-O'kuru et al., 2015) . The 1 H-NMR and 13 C-NMR analysis were shown in Table 3 exhibit characteristics of triglyceride (TG) (Sarpal et al., 2016) . Table 3 confirmed the triglyceride characteristics as we can compared to previous report. Sarpal et al., (2016) report the 1 H-NMR signals at 4.38 to 5.2 ppm were assigned to protons of OCH2 and OCH ester groups and 13 C-NMR spectra display OCH2 (sn-1, 3) and OCH (sn-2) of triglyceride at 62.05 and 68.87 ppm. The unsaturated carbons (-CH=CH) signal were between 126-132 ppm. The appearance of characteristics signals at 127.12 and 132.06 ppm due to C18:3 (Sarpal et al., 2016 ) while here we observed the single signal both at 127.25 and 132.11 ppm. Mass spectrum and molecule fragmentation of triglyceride identified by LC-MS/MS in the n-hexane extract of M. vitifolia. This compound namely triglyceride 1 is second most abundance compound with retention time at 11.8618 min in the sample 1 based on the chromatogram in Figure 1 . An unusual feature of the mass spectra of triglycerides is an ion corresponding to the loss of water, the [M+H-18] + peak. This fragmentation is unique among the mass spectra of esters (Hites, 1975) . The [M+H] + peak of Triglyceride 1 is m/z 855.55226. We can calculate the chemical formula of triglyceride 1 is C58H92O6. The mass fragmentation of TG show specific fragment for the loss of RCOOH (fatty acid fragment) (Duval et al., 2016) . Figure 5 shows the loss mass fragment of linolenic or trichosanic acid (C18H30O2) which means that the triglyceride 1 comprised of linolenoyl or trichosanoyl (C18:3) unit as previously observed in 13 C-NMR signal.
Chemicals Identification by LC-MS/MS in Methanol Extract of M. vitifolia The LC-MS/MS analysis of methanol extract of M. vitifolia has been conducted. The chromatogram results for sample 4 is shown in Figure 6 . Table 4 shows all the proposed compound that identified by LC-MS/MS analysis in methanol extract of M. vitifolia (sample 4). Blank chromatogram has been showed in Figure 3 .
Chemicals Identification by GC-MS in Methanol Extract of M. vitifolia The GC-MS analysis of methanol extract of M. vitifolia has been conducted. Table 5 shows the compounds that identified by GC-MS analysis in methanol extract of M. vitifolia (sample 2). Identification of the substances used Library NIST17.L. The two most abundance compounds in this GC-MS analysis of methanol extract of M. vitifolia are (Z)-15octadecenoic acid methyl ester and n-hexadecanoic acid.
Chemicals Identification by LC-MS/MS in Metanol Extract of B. pilosa The LC-MS/MS analysis of methanol extract of B. pilosa has been conducted. The chromatogram results for sample 5 is shown in Figure 7 . Table 5 shows all the proposed compound that identified by LC-MS/MS analysis in methanol extract of B. pilosa (sample 5). Blank chromatogram has been showed in Figure 3 .
Chemicals Identification by GC-MS in Methanol Extract of B. pilosa The GC-MS analysis of methanol extract of B. pilosa has been conducted. Table 7 shows all the compounds that identified by GC-MS analysis in methanol extract of B. pilosa (sample 3). Identification of the substances used Library NIST17.L. The two most abundance compounds in this GC-MS analysis of methanol extract of B. pilosa are (E)-9-Octadecenoic acid and n-Hexadecanoic acid. Histamine Inhibiting Test Result Histamine inhibition test was a quantitative test to determine the concentration of histamine in the flesh of fish using spectrophotometer UV-Vis. For Histamine standard curve (Supplement Data), the variety of standard concentrations were made, and absorbances were measured at a wavelength of 496 nm. The increasing of histamine content in the meat of Frigate Tuna after treatment with various concentration of extracts and 4-hydroxybenzoic acid (4-HBA) has been shown in Figure 8 . The histamine content was calculated using 4-diazoniobenzenesulfonate reagent and determined by spectrophotometer UV-Visible at wavelength of 496 nm in duplet (Figure 9 ). We used 4-hydroxybenzoic acid (4-HBA) control standard because 4-HBA is known as antimicrobial substances capable of inhibit most of the Grampositive and some Gram-negative bacteria (Cho et al., 1998; Kosová et al., 2015) . The weakness of using 4-HBA is the low solubility in water. We had to increase the temperature to dissolved 4-HBA, with vigorous stirring. Bacterial activity is the primary cause of histamine production in the fish. The average histamine concentration formed after treatment with M. vitifolia extract is smaller compared other treatments especially 4-HBA treatment. This means that M. vitifolia extract is better histamine formation inhibitor compared to Bidens pilosa extract or 4-HBA. This result has similar conclusion to previous reports (Bartolome et al., 2013; Hasanah et al., 2020; Xuan & Khanh, 2016) that polar extract of both plant's part could strongly inhibit the growth of many bacteria. Some lipids have potential as antimicrobial. The fatty acid is the precursors of triglyceride formation in cells. Chemicals like n-hexadecanoic acid, (E)-9octadecenoic acid, hexadecanoic acid methyl ester, spinasterol have potent antimicrobial activities as shown in Table 8 and 9. The activity of histidine decarboxylase is also important in the formation of histamine in fish. Potent inhibitor of histidine decarboxylase is 2-hydroxy-5carbomethoxybenzyloxyamine (Huszti & Sourkes, 1975) . This chemical was a derivate of 4hydroxybenzoic acid, so we supposed to observe any inhibition activity of 4-HBA. Histamine formation in fish after 30 min treatment with 4-HBA is around 40 -51 mg / 100 g of fish. This value is more than 5 times bigger compared to treatment with the extracts, which means 4-HBA has less inhibitory activity. The European Commission Regulation set the maximum histamine content in fresh fish should equal to 20 mg per 100 g of fish. At this concentration no adverse effect caused by histamine to average human (Visciano et al., 2014) . We observed that histamine concentration in fish was less than 10 mg / 100 g of fish after 30 min treatment with M. vitifolia and B. pilosa extracts. This value is smaller than other report of Tamarind extract (20% b/v) effect to histamine content in bullet Tuna after 30 min treatment by Hattu et al., (2015) which is 17.693 mg/100 g of fish. The smaller the histamine content in fish means the higher the inhibitory effect the extract has. This data shows the preliminary indication of high potential of both extract as preserver of fish products. We also observed the small increasing of histamine as the M. vitifolia extract concentration increased. This phenomenon possibly due to the amount of histamine derivate that detected by GC-MS in the extract that has influenced UV-Vis measurements.
Bioactivity of The Identified Substances The first aim of this study is to identify active substances in the extract of M. vitifolia dan B. pilosa using LC-MS/MS and GC-MS analysis. In LC-MS/MS analysis, chemicals identification based on mass spectrum, references, and exact neutral mass (Da) of the substance obtained from mass spectrum. The GC-MS analysis using library NIST.17.L to observe the highest similarity score of mass fragments spectrum. We identified 27 chemicals in M. vitifolia extract and 14 chemicals in B. pilosa extract. Some of those substances have biological activities as shown in Table 8 and 9. Table 8 . Bioactivity of some substances identified in B. pilosa extract
Name of compound Bioactivity Hexadecanoic acid, methyl ester Anti-inflammatory (Igwe, 2014) , antibacterial (Shaaban et al., 2021) , antifungal (Abubacker & Deepalakshmi, 2013 ) n-Hexadecanoic acid Anti-inflammatory (Gopu et al., 2021) , antibacterial (Johannes et al., 2016 ) (E)-9-Octadecenoic acid Antibacterial and antifungal (Ghavam et al., 2021 ) Octadecanoic acid Antibacterial (Pu et al., 2010) 5,7,2',5'-tetrahydroxy-flavone Anticancer (Lian et al., 2021; Sonoda et al., 2004 ) N1,N5,N10-tricoumaroyl spermidine Hepatoprotective (Zhou et al., 2021) , antioxidant (Negri et al., 2011) Reduce platelet aggregation in human (Takenaga et al., 1988 ) Pheophorbide a Potent endothelin receptor antagonist (Ohshima et al., 1994) , antiviral, anti-inflammatory, antioxidant and anti-proliferative effects in several human cancer cell (Saide et al., 2020)
Methyl pheophorbide Antioxidative and anticarcinogenic (Das et al., 2016) CONCLUSIONS By using LC-MS/MS and GC-MS, 27 chemicals in M. vitifolia extract and 14 chemicals in B. pilosa extract have been detected and identified. A triglyceride has been isolated and identified by using FTIR, H-NMR and C-NMR supported by LC-MS/MS data. Histamine concentration in fish was less than 10 mg / 100 g of fish after 30 min treatment with M. vitifolia and B. pilosa extracts. Histamine formation in fish after 30 min treatment with 4-hydroxybenzoic acid is around 40 -51 mg / 100 g of fish. This data shows the preliminary indication of high potential of both extract as preserver of fish products. Δ[Histamine](mg/100 g)=[Histamine]t -[Histamine]0 (1) Where: Δ[Histamine] = the increasing of histamine concentration in mg/100 g of fish meat, [Histamine]t = histamine concentration after 30 minutes immersing treatment, [Histamine]0 = histamine concentration at 0 minutes treatment.
Figure 1 . 1 Figure 1. Chromatogram LC-MS/MS of n-hexane extract of M. vitifolia (sample 1) in comparison with chromatogram of the blank.
Figure 3 . 3 Figure 3. Chromatogram LC-MS/MS of n-hexane extract of M. vitifolia (sample 6a) in comparison with chromatogram of the blank.
Figure 4 . 4 Figure 4. FTIR analysis of isolated fraction 1 of M. vitifolia
Figure 5 . 5 Figure 5. MS spectrum of triglyceride 1 identified in n-hexane extract of M. vitifolia
Figure 6 . 6 Figure 6. Chromatogram LC-MS/MS of methanol extract of M. vitifolia (sample 4).
Figure 7 . 7 Figure 7. Chromatogram LC-MS/MS of methanol extract of B. pilosa (sample 5)
Figure 8 . 8 Figure 8. The increasing of histamine concentration (mg/100 g) in the fish meat after 30 minutes treatment with extracts and 4-hydroxybenzoic acid solution.
Figure 9 . 9 Figure 9. The immersing of fish meat in 4-hydroxybenzoic acid solution (A), in the extract of B. pilosa (B), and in the extract of M. vitifolia (C).
Table 1 . 1 No RT (Min) Observed Neutral Mass Adducts Mass Proposed compound Ref. 1 7.35 2 7.60 (m/z) 197.12 415.16 (Da) 196.10994 414.15796 H + H + Error 0.8 Digiprolactone (mDa) -1.3 Gamma-sitosterol Library Instrument (Tahya & Karnelasatri, 2021) 3 9.71 181.12 180.11503 H + 1.0 Dihydroactinidiolide PubChem CID 27209 4 9.93 277.22 276.20893 H + 0.6 Stearidonic acid Library Instrument 5 10.09 625.27 624.25706 H + 1.4 Diglyceride + H2O (Duval et al., 2016) 6 10.14 593.28 592.26723 H + 1.4 Pheophorbide a PubChem CID 101690780 7 11.13 935.55 934.54424 H + 1.4 Epigallocatechin gallate PubChem CID 4,4'-dipalmitate 76958273 8 11.29 405.37 382.38108 Na + 2.9 Pentacosanoic acid Library Instrument 9 11.44 413.38 412.37052 H + 0.4 Spinasterol Library Instrument 10 11.86 903.56 902.55441 H + 1.3 Polyunsaturated (Duval et al., 2016) Triglyceride (C58H92O6) (PubChem CID + H2O 131765888) 11 11.97 887.57 886.55950 H + 1.1 Polyunsaturated (Duval et al., 2016) Triglyceride (C57H88O6) (PubChem CID + H2O 131765838) 12 12.15 871.57 870.56458 H + 0.8 Polyunsaturated (Duval et al., 2016) Triglyceride (C57H90O6) (PubChem CID 131765968) The proposed compounds that are identified by LC-MS/MS in n-hexane extract of M. vitifolia from sample 1.
Table 2 . 2 The proposed compounds that are identified by LC-MS/MS in n-hexane extract of M. vitifolia from sample 6a. Hexane Extract of M. vitifolia TLC preparative result of the n-hexane extract of M. vitifolia shows only one spot under UV light. This isolation proses has high possibility to isolate a compound as only one spot observed in TLC plat as shown in Figure No RT Observed Neutral Mass Adducts Mass Error Proposed Ref. (Min) (m/z) (Da) (mDa) compound 1 10.03 609.27 586.27780 H + 4.5 Azedarachin C Library Instrument 2 7.34 277.22 276.20893 H + -0.1 Stearidonic acid Library Instrument 3 12.91 429.37 428.36543 H + 0.2 Stigmastan-3,6- Library dione Instrument 4 9.47 279.23 278.22458 H + 0.6 Trichosanic acid Library Instrument Isolation and Identification of Triglycerides from n-
Table 3 . 3 Chemicals shift of 1 H-NMR and 1 C-NMR of isolated triglyceride from M vitifolia extract. δ H (ppm), multiplicity δ C (ppm) Corresponding Functional group 4.1246 -4.1602, dd 61.3492 CH2-O (Glyceryl) 4.2748 -4.3071, dd 62.2476 CH2-O (Glyceryl) 4.5772 -4.5914, d 69.0072 CH-O (Glyceryl) - 173.0053 -173.4249 O-C=O (Ester carbonyls) 5.0452 -5.4067, m 124.1585 -132.1081 HC=CH2-(olefinic) 6.3497 -6.4065, m 113.2026 -118.2671 HC=C-C=C 2.2774 -2.3298, m 34.1695 -34.5569 CH-C=O 2.1916 -2.3298, m 25.6727 -25.8548 CH-C=C 2.7544 -2.8041, m 26.7387 -26. 9081 C=C-CH-C=C 0.8704 -0.8911, m 14.2358 -14.4378 Terminal -CH3 1.2027 -1.2507, m 29.5007 -29.8554 -(CH2)n-
Table 4 . 4 The five most abundance proposed compounds that are identified by LC-MS/MS in methanol extract of M. vitifolia from sample 4. No Observed m/z Neutral Mass Observed Proposed compound Ref. (Da) RT (min) 1 609.27 586.27780 10.03 Azedarachin C Library Instrument 2 279.23 278.22458 9.47 Trichosanic acid Library Instrument 3 593.28 592.26857 10.23 Pheophorbide A PubChem CID 253193 4 607.29 606.28422 11 Methyl pheophorbide PubChem CID 73074 5 653.30 652.28970 10.9 Pheopurpurin 7, PubChem trimethyl ester CID 135441796
Table 5 . 5 Identified compounds in methanol extract of M. vitifolia using GC-MS analysis. No Name of compound RT (Min) % Area SI (%) Formula 1 2-(tert.- 7.495 4.638 70.03 C21H26O2Si Butyldimethylsilyl)oxybenzylidene acetophenone 2 Hexadecanoic acid, methyl ester 32.098 6.348 92.03 C17H34O2 3 n-Hexadecanoic acid 32.774 21.385 93.08 C16H32O2 4 9-Octadecenoic acid (Z)-, methyl 35.475 7.727 83.75 C19H36O2 ester 5 2-Hexenal, (E) 35.694 3.286 82.5 C6H10O 6 Histamine, N-trifluoroacetyl 36.068 1.435 60.2 C7H8F3N3O 7 (Z)-15-Octadecenoic acid methyl 36.359 50.364 67.52 C19H36O2 ester 8 2-tert-Butylphenol, tert- 50.735 4.019 53.29 C16H28OSi butyldimethylsilyl ether 9 3-Butoxy-1,1,1,5,5,5-hexamethyl-3- 56.088 0.422 55.46 C13H36O4Si4 (trimethylsiloxy)trisiloxane
Table 6 . 6 The five most abundance proposed compounds that are identified by LC-MS/MS in methanol extract of B. pilosa from sample 5. No Observed m/z Neutral Mass Observed Proposed compound Ref. (Da) RT (Min) 1 287.05 286.04774 4.25 5,7,2',5'-Tetrahydroxy-flavone Library Instrument 2 271.06 270.05282 4.67 Digitopurpone Library Instrument 3 787.37 786.36286 4.79 N1,N5,N10,N14-Tetra-Trans-P- PubChem CID Coumaroylspermine 9810941 4 584.28 583.26824 4.5 N1,N5,N10-tricoumaroyl PubChem CID spermidine 14777879 5 185.10 184.08882 6.26 Isoquinaldonitrile, 3-methyl-, 2- GC-MS oxide confirm
Table 7 . 7 Identified compounds in methanol extract of B. pilosa using GC-MS analysis No Name of compound RT (Min) % Area SI (%) Formula 1 1,1,1,3,5,5,5- 0.165 1.466 60.32 C7H22O2Si3 Heptamethyltrisiloxane 2 Isoquinaldonitrile, 3-methyl-, 2- 28.59 0.703 87.83 C 11 H 8 N 2 O oxide 3 Hexadecanoic acid, methyl ester 32.097 1.222 91.5 C17H34O2 4 n-Hexadecanoic acid 32.851 21.746 97.28 C16H32O2 5 (Z)-15-Octadecenoic acid methyl 35.475 1.559 84.08 C19H36O2 ester 6 9-Octadecenoic acid, (E)- 36.264 68.570 71.29 C18H34O2 7 Octadecanoic acid 36.602 2.630 65.05 C18H36O2 8 Glycidyl palmitate 38.917 0.727 63.97 C19H36O3 9 9-Octadecenoic acid (Z)-, 41.831 1.377 54.34 C 21 H 38 O 3 oxiranylmethyl ester
Table 9 . 9 Bioactivity of some substances identified in M. vitifolia extract Name of compound Biological activity Spinasterol Antibacterial activity (Yang et al., 2017), anti- inflammatory, antidepressant, antioxidant and antinociceptive effects (Brusco et al., 2017), anticancer (Meneses-Sagrero et al., 2017), antifungal, and aphicidal (Ahmed et al., 2022) Stigmastan-3,6-dione Apoptosis induction of cancer cells (Duarte et al., 2009) Trichosanic acid
|
10.18632/oncotarget.15065
|
cc-by
| null | null |
openalex
|
Recent studies have demonstrated that P-glycoprotein (P-gp) expression impairs DNA interstrand cross-linking agent-induced DNA repair efficiency in multidrugresistant (MDR) cells. To date, the detailed molecular mechanisms underlying how P-gp interferes with Src activation and subsequent DNA repair activity remain unclear. In this study, we determined that the C-terminal Src kinase-binding protein (Cbp) signaling pathway involved in the negative control of Src activation is enhanced in MDR cells. We also demonstrated that cells that ectopically express P-gp exhibit reduced activation of DNA damage response regulators, such as ATM, Chk2, Braca1 and Nbs1 and hence attenuated DNA double-strand break repair capacity and become more susceptible than vector control cells to DNA interstrand cross-linking (ICL) agents. Moreover, we demonstrated that P-gp can not only interact with Cbp and Src but also enhance the formation of inhibitory C-terminal Src kinase (Csk)-Cbp complexes that reduce phosphorylation of the Src activation residue Y416 and increase phosphorylation of the Src negative regulatory residue Y527. Notably, suppression of Cbp expression in MDR cells restores cisplatin-induced Src activation, improves DNA repair capacity, and increases resistance to ICL agents. Ectopic expression of Cbp attenuates cisplatin-induced Src activation and increases the susceptibility of cells to ICL agents. Together, the current results indicate that P-gp inhibits DNA repair activity by modulating Src activation via Cbp-Csk-Src cascade. These results suggest that DNA ICL agents are likely to have therapeutic potential against MDR cells with P-gp-overexpression.INTRODUCTION Multidrug resistance (MDR) is a significant obstacle to the success of chemotherapy in cancer patients . Although MDR may be attributed to various mechanisms, it is often associated with increased expression of ATPbinding cassette (ABC) transporter family members, which extrude anticancer drugs out of cells [2, 3] . The MDR1 gene product, P-glycoprotein (P-gp), is one of the most well-known ABC transporters. ABC transporters expel a broad range of bioactive chemicals , including various anticancer drugs, such as vinblastine, vincristine, doxorubicin and paclitaxel [5, 6] . Thus, overexpression of P-gp in tumor tissues is a prognostic indicator associated with poor response to chemotherapy and poor clinical outcome . Numerous agents have been identified or developed to modify, modulate, or reverse the P-gpmediated MDR phenotype [1, 10, 11] . However, most of those agents were terminated during clinical trials because of their toxicities or unexpected outcomes . Therefore, developing novel agents against P-gp and targeting alternative mechanisms that sensitize MDR cells to therapeutic agents may represent new paths toward overcoming MDR [11, 13] .
Research Paper Alternatively, numerous studies have shown that cancer cells with acquired MDR or ectopically expressed P-gp have increased sensitivity to DNA-damaging agents, including cisplatin [14, 15] . Our previous study has also found that P-gp overexpression attenuates DNA repair in MDR cells damaged by DNA interstrand cross-linking (ICL) agents . However, studies investigating how P-gp interferes with DNA repair are limited. We have previously revealed that Src activation by DNA-damaging agents is significantly reduced by P-gp overexpression in MDR cells . Because Src signaling plays crucial roles in the regulation of the DNA damage response (DDR) , our study suggests that P-gp interferes with Src activation. Src, the first identified oncogene encoding a non-receptor tyrosine kinase, plays pivotal roles in coordinating diverse cellular responses involved in differentiation, adhesion and migration . Src is overexpressed and activated in various human cancers , suggesting its role in tumor progression. Src also participates in controlling several parameters of cancer metastasis and drug resistance [21, 22] . Several reports have identified associations between Src and resistance to irradiation, cisplatin, and paclitaxel [17, 23, 24] . Src activation-induced drug resistance is likely because of the activation of DNA-PK and enhancement of DNA double-strand break repair . In addition, Src causes the dissociation of cyclindependent kinase 2 from cyclin A and induces S-phase arrest, thereby enhancing the repair of etoposide-induced DNA damage . Therefore, targeting Src offers a novel therapeutic intervention strategy against cancer, particularly in the augmentation of chemosensitivity . Oncogenic activation of Src requires phosphorylation at tyrosine 416 (pSrc Y416 ) of the catalytic domain, whereas the enzymatic activity of Src is blocked when the tyrosine 527 of the C-terminal regulatory element is phosphorylated (pSrc Y527 ) . Phosphorylation of the C-terminal regulatory Y527 on Src is catalyzed by C-terminal Src kinase (Csk), a unique regulatory tyrosine kinase of the Src family . Src is anchored to specific lipid-raft membrane domains via N-termini, whereas Csk, a cytoplasmic protein, is recruited via adaptor proteins to the membrane and inhibits Src . Among several Csk-binding proteins , Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid-enriched membrane, is exclusively localized to lipid rafts and recruits Csk to efficiently inactivate Src [36, 37] . Therefore, Cbp is known as a transmembrane adaptor protein of Csk. Previous studies have proposed that Cbp acts as a suppressor of Srcmediated cell migration , tissue repair , and tumor progression . A recent study has also shown that P-gp plays a special role in Cbp recruitment . Accordingly, we hypothesized that Cbp is a critical component involved in the P-gp-mediated regulation of Src in DNA-damaging agent-induced DNA repair. In this study, we conducted experiments to investigate whether P-gp mediates Cbp-Csk-dependent signaling to attenuate Src activation, thereby reducing DNA repair activity in MDR cells.
RESULTS
Ectopic expression of P-glycoprotein inhibits cisplatin-induced activation of Src at Y416 While we previously demonstrated that DNA damage significantly induces Src activation in parental KB cells but not in P-gp-overexpressing KBvin10 cells , we first confirmed that KBvin10 cells were more susceptible than KB cells to DNA cross-linking agents, such as cisplatin and BO-1922, a synthesized DNA ICL derivative of indolizino [6,7-b] indole (compound 18a in our previous report) (Figure 1A ). The IC 50 values of KB cells to cisplatin and BO-1922 were 3.0 and 1.6 folds higher than those of KBvin10 cells, respectively. We further observed that cisplatin treatment markedly enhanced activated Src (pSrc Y416 ) but significantly reduced inactivated Src (pSrc Y527 ) in KB cells (Figure 1B ). On the contrary, pSrc Y527 was enhanced whereas pSrc Y416 remained at a relatively low level in cisplatin-treated KBvin10 cells (Figure 1B ). Moreover, we observed significantly increased EGFR phosphorylation at residue Y 845 (pEGFR Y845 ), known as Src kinase specific phosphorylation site , in KB cells but not in KBvin10 cells treated with cisplatin (Figure 1B ). Total EGFR expression was not changed in either KB or KBvin10 cells after cisplatin treatment (Figure 1B ), implying that Src activation in cisplatin treated KBvin10 cells was attenuated by overexpressed P-gp. MDR cells are generally established by long-term drug selection . To further understand how P-gp attenuates DNA-damaging agent-induced Src activation, we ectopically expressed P-gp in Paca-S1 cells and established stable clones (Paca-S1-P1 and Paca-S1-P7) by culturing them in the medium containing vincristine. Because Paca-S1-P1 cells express higher levels of P-gp than Paca-S1-P7 cells (Figure 2A ), they were chosen for this study. We first assessed the acquired resistance of Paca-S1-P1 cells to MDR drugs, such as vincristine, vinblastine, doxorubicin, etoposide, and paclitaxel. As summarized in Table 1 , Paca-S1-P1 cells were crossresistant to all of these MDR drugs. However, Paca-S1-P1 cells were more susceptible than Paca-S1-V cells to DNA cross-linking agents, such as cisplatin, melphalan, carboplatin, and BO-1922. These results were consistent to our previous findings . Furthermore, we showed that treatment of Paca-S1-V cells with 50 μM cisplatin for 1 h and followed by incubation in drug-free medium resulted in increased pSrc Y416 but decreased pSrc Y527 in a time-dependent manner (Figure 2B ). The relative intensity of pSrc Y416 and pSrc Y527 at 8 h was 1.85 ± 0.03 (n = 3) and 0.66 ± 0.01 (n = 3) in Paca-S1-V cells, respectively. However, no notable change was observed in Paca- S1-P1 cells treated with cisplatin. We further confirmed these findings by treatment of Paca-S1-V cells or Paca-S1-P1 cells with various concentrations of cisplatin for 4 h. As shown in Figure 2C , activated pSrc Y416 was increased whereas inactivated pSrc Y527 decreased in a dose-dependent manner in Paca-S1-V cells but not in Paca-S1-P1 cells. The relative intensity of pSrc Y416 and pSrc Y527 at 100 μM to control was 2.27 ± 0.04 (n = 4) and 0.53 ± 0.04 (n = 4) in Paca-S1-V cells, respectively. However, there was no change in Paca-S1-P1 cells. In addition, we also observed that cisplatin treatment resulted in dose-dependent increase of pEGFR Y845 in Paca-S1-V cells but dose-dependent decrease in Paca-S1-P cells. Since KBvin10 and Paca-S1-P1 cells were acquired by selection in medium containing vincristine, we performed similar experiments using KB cells that were transiently expressed P-gp without drug selection. As shown in Supplementary Figure 1 , similar results were observed, suggesting that P-gp indeed played certain role on attenuating the Src activation. These results similar to those observed in KBvin10 cells further implicated that P-gp may contribute to the resistance of MDR drugs by attenuation of DNA damaging agent induced Src activation.
Ectopic expression of P-glycoprotein suppresses DNA damage response Since we have shown increased susceptibility to DNA crosslinking agents in P-gp overexpressing cells, we then adopted phosphorylated histone H2AX (γH2AX) as DNA damage marker to further confirm the interference of P-gp in Paca-S1 cells transfected with a P-gp-expressing vector. Two stable P-gp expressing cell lines, Paca-S1-P1 and -P7, were established and maintained in medium containing 10 nM vincristine. Paca-S1-V cells were transfected with expression vector and served as a control. The protein levels of P-gp were analyzed by western blotting. (B and C) Time-and dose-dependent modulation of pSrc Y416 and pSrc Y527 in Paca-S1-V and -P1 cells, respectively. As described in Figure 1 , Paca-S1-V and Paca-S1-P1 cells were treated either with 50 μM cisplatin for 1 h, washed, and incubated in the fresh medium for various time periods as indicated (B) or various concentrations for 1 h, washed, and incubated for 4 h (C). The protein levels of total Src, pSrc Y416 , pSrc Y527 , EGFR, and pEGFR Y845 were analyzed by western blotting. of DNA repair in P-gp overexpressing cells. As shown in Figure 3A , γH2AX was significantly increased at 24 h and gradually declined at 48 and 72 h in Paca-S1-V, indicating that the damaged DNA was gradually repaired. However, the levels of γH2AX were constantly maintained in Paca-S1-P1 cells up to 72 h, implying no significant DNA repair in Paca-S1-P1 cells. Similar results were observed in Paca-S1-V and Paca-S1-P1 cells treated with BO-1922, which is a potent agent to induce DNA interstrand crosslinks (Supplementary Figure 2 ). These results implicated that attenuated Src activation by overexpressed P-gp may interfere with DDR. As shown in Figure 3B , we did not observed the change of protein levels of several proteins involved in DDR, such as ATM, Chk2, Brca1, Nbs1, Mre11, Rad50, Rad51, and FANCD2, in cisplatin treated cells either with P-gp overexpression (Paca-S1-P1 and KBvin10 cells) or without (Paca-S1-V cells and KB cells). However, we found that in response to cisplatin treatment the phosphorylated ATM (pATM S1981 ), Chk2 (pChk2 T68 ), Brca1 (pBrca1 S1524 ), and Nbs1 (pNbs1 S343 ) were increased in a time-dependent manner in Paca-S1-V cells and KB cells, respectively. In cisplatin-treated Paca-S1-P1 and KBvin10 cells, the levels of phosphorylation of these proteins were apparently less than that in Paca-S1-V cells and KB cells, respectively. These results supported that P-gp attenuated Src activation and subsequently prevented the activation of DDR signaling in cells treated with DNA damaging agents. Consequently, P-gp overexpressing cells were more susceptible to DNA cross-linking agents compared to their parental cells.
P-glycoprotein interacts with Src, Cbp, and Csk Src is negatively regulated by Csk, which phosphorylates Src at Y527 . While Csk was recruited to the membrane by the Src suppressor Cbp , P-gp was reported to recruit Cbp to the membrane . We therefore investigated the role of Csk and Cbp in P-gp resulted in suppression of DNA damage induced Src activation. We first observed that P-gp overexpression did not affect Csk protein levels (Figure 4A ). However, Cbp protein levels (Figure 4A ) and mRNA levels (Figure 4B ) were significantly enhanced in KBvin10 and Paca-S1-P1 cells compared to their parental KB and Paca-S1-V cells, respectively. We further observed a slow degradation of Cbp in KBvin10 cells compared with KB cells after treatment with cycloheximide (CHX) (Figure 4C ). The estimated half-life of Cbp in KB cells was approximately 2 h, whereas no apparent Cbp degradation was observed in KBvin10 cells within 4 h. These results implied that P-gp not only enhances the expression of Cbp but also prolongs the stability of Cbp. To explore the underlying mechanism through which P-gp attenuates Src activation, we examined the interaction between P-gp, Src, and Cbp by reciprocal immunoprecipitation assays using antibodies against P-gp, Src, and Cbp, respectively. As shown in Figure 5A , we confirmed the interaction between Src and Cbp. However, using antibody against one of these proteins, we observed that the other 2 proteins were co-precipiated in lysates of KBvin10 cells. These results revealed a direct interaction between P-gp, Cbp and Src in P-gp overexpressing KBvin10 The co-localization of P-gp and Src as well as P-gp and Cbp on the plasma membrane was further confirmed by immunofluorescence analysis in KBvin10 cells (Figure 5B ). Similar results were found in Paca-S1 cells (Supplementary Figure 3 ). In addition, a large portion of Src and Cbp were localized in the cytosol. Intriguingly, the interaction of P-gp and Csk was limited in KBvin10 cells without exposure to DNA-damaging agents but significantly increased upon cisplatin treatment (Figure 6 ). However, the interaction of Cbp and Csk was not affected by cisplatin treatment. Accordingly, we may infer that upon cisplatin treatment P-gp enhances the recruitment of Csk from the cytosol to the cell membrane, where Csk phosphorylates Src at Y527 and attenuates Src activation.
P-glycoprotein and Cbp silencing restores cisplatin-induced activation of Src at Y416 and DNA repair capacity in MDR cells To further reveal the roles of P-gp and Cbp in the suppression of cisplatin-induced Src activation, we established P-gp-and Cbp silenced KBvin10 cells by infecting the cells with P-gp or Cbp lentiviral shRNA, respectively. KBvin10-shLuc cells infected with lentiviral luciferase shRNA were used as a control. Reduced P-gp and Cbp protein levels in KBvin10-shP-gp and KBvin10-shCbp compared to KBvin10-shLuc cells was demonstrated by western blotting analysis (Figure 7A ). We also confirmed increased resistance to cisplatin in P-gp or Cbp silenced KBvin10 cells (Figure 7B ). As described above, we inferred that P-gp restrained Cbp turnover, resulting in its accumulation and hence suppression of DNA damage-induced Src activation. We further demonstrated that the DNA damage marker γH2AX accumulated in KBvin10-shLuc cells treated with cisplatin up to 48 h and then declined (Figure 7C ). However, no significant increase in γH2AX was observed in cisplatintreated KBvin10-shCbp cells (Figure 7C ), implying that cisplatin-induced DNA damages were rapidly rescued and repaired in KBvin10-shCbp cells compared with KBvin10-shLuc cells. To confirm this finding, we adopted a modified comet assay to compare the repair activity of DNA ICLs in KBvin10-shLuc and KBvin10-shCbp cells. As shown in Figure 7D , Cbp silencing accelerated the repair of DNA ICLs. Moreover, the effects of cisplatin treatment on Src activation in KBvin10-shLuc, KBvin10-shP-gp, and KBvin10-shCbp cells were examined by determining the levels of pSrc Y416 and pSrc Y527 . Similar to the parental KBvin10 cells, no significant increase of pSrc Y416 was observed in the cisplatin-treated vector control KBvin10-shLuc cells (Figure 8A ). Alternatively, pSrc Y527 was significantly increased in cisplatin-treated control KBvin10-shLuc (Figure 8A ). However, activated pSrc Y416 was significantly increased by cisplatin in both KBvin10-shP-gp cells (Figure 8B ) and Cbp-silenced KBvin10-shCbp cells (Figure 8C ). Consistently, the protein levels of inactivated pSrc Y527 were reduced in cisplatin treated KBvin10-shP-gp cells (Figure 8B ) and KBvin10-shCbp cells (Figure 8C ). These results further support that P-gp and Cbp may serve as negative regulators of Src. Their overexpression in MDR cells was likely to sensitize the MDR cells to DNA cross-linking agents.
Ectopic expression of Cbp inhibits cisplatininduced activation of Src in KB cells To further verify the inhibitory role of Cbp in DNAdamaging agent-induced Src activation, we ectopically expressed Cbp in KB cells and established stable clones (KB-AcGFP-Cbp) (Figure 9A ). We observed that KB-AcGFP-Cbp cells were more susceptible to cisplatin than KB and control KB-AcGFP cells (Figure 9B ). Furthermore, cisplatin treatment failed to induce pSrc Y416 but significantly increased pSrc Y527 in KB-AcGFP-Cbp cells (Figure 9C ), supporting the inhibitory role of Cbp in cisplatin-induced Src activation. We further performed an in vivo assay to evaluate the therapeutic activity of cisplatin against KBvin10 cells with differential expression of Cbp. As shown in Figure 9D , the KBvin10-shLuc and KBvin10-shCbp xenografts were treated in situ with normal saline or cisplatin at 4 mg/kg. At day 10, cisplatin suppressed the growth of the KBvin10-shLuc and KBvin10-shCbp xenografts by 80.5% and 41.1%, respectively, suggesting that silencing of Cbp resulted in increased cisplatin resistance. Similarly, cisplatin treatment reduced the tumor growth of KB-GFP and KB-Cbp xenografts by 54.6% and 80.7%, respectively (Figure 9E ), confirming that enhanced expression of Cbp sensitizes the cells to cisplatin. Together, these results indicate that P-gp interacts with Cbp and Src to recruit Csk to attenuate Src activation upon DNA injury, a crucial signal for activation of the DNA repair machinery.
DISCUSSION Although P-gp acts as an efflux pump responsible for preventing the delivery of various chemotherapeutic agents to cancer cells, several reports have demonstrated that increased P-gp levels enhance the sensitivity of MDR cancer cells to various DNA-damaging agents, such as cisplatin, bis-chloroethylnitrosourea, and gemcitabine Figure 6 : Association of P-gp with Csk in cisplatin-treated KBvin10 cells. KBvin10 cells were treated with or without cisplatin at 20 μM for 1 h, washed, and then incubated with fresh medium for 2 h. Cell lysates were immunoprecipitated with antibodies against P-gp or Cbp and followed by western blotting with anti-P-gp, anti-Csk, or anti-Cbp antibodies, respectively. Normal mouse immunoglobulin (IgG) was included as a negative control and 1/10 of whole cell lysates (WCL) used for immunoprecipitation was included as loading control. [14, 15, 46] . Several possible explanations have been reported to account for the toxicity of P-gp-potentiated compounds, including generation of reactive oxygen species through ATP hydrolysis, altered drug metabolism, and membrane perturbation [47, 48] . We have previously shown that overexpressed P-gp in MDR cells attenuates the Src-activated repair activity by DNA ICLs and contributes to the susceptibility of MDR cells to DNA ICL agents . In this report, we revealed mechanistic insights regarding how P-gp interferes with Src activation and DNA repair activity. Our results first demonstrated that the expression levels of P-gp were inversely associated with Src activation and repair of cisplatin-induced DNA ICLs. Second, we noticed that P-gp overexpression attenuated the DDR upon treatment with DNA damaging agents. Third, we observed significantly increased expression of Cbp in P-gp-overexpressing cells, in which P-gp interacts with Cbp and prolongs its stability. Fourth, upon cisplatin treatment, we revealed an interaction between P-gp and Csk in KBvin10 cells in which Cbp levels were enhanced. Fifth, the repair of cisplatin-induced ICLs and Src activation were suppressed by ectopically expressed Cbp, whereas Cbp silencing enhanced cisplatin-induced Src activation and increased the resistance of MDR cells to DNA ICL agents. Together, our present results revealed that overexpressed P-gp in MDR cells mediates through a Cbp-dependent mechanism to attenuate and Src activation and DNA repair activity. Src and other Src family kinases are overexpressed and participate in numerous signaling pathways in various human cancers . Activated Src has also been shown to cause cancer cells to be resistant to various chemotherapeutic agents and targeted therapeutics [50, 51] . Actually, numerous reports have demonstrated that Src activation is involved in drug resistance by enhancing DNA repair . Our present results showing that cisplatin treatment significantly increased the levels of pSrc Y416 (activated Src) as well as pEGFR Y845 in KB cells but not in P-gp overexpressed KBvin10 cells. The phosphorylation of EGFR at Y845 was mediated by activated Src and involved in regulation of DNArepair . Furthermore, we found that overexpressed P-gp, either in acquired MDR cells or in ectopically expressed cells, interfered with cisplatin-induced phosphorylation of ATM, Chk2, Brca1 and Nbs1 that have been known as major regulators of DDR, which activate different DNA repair processes, including homologous recombination repair, non-homologous end joining repair . These results supported that suppression of DNA-damaging agent-induced Src activation by P-gp is likely to inhibit DDR in MDR cells. This may be a reason to explain why MDR cells are more susceptible to cisplatin or other DNA ICL agents than the parental cells. Uncovering the mechanisms of Src regulation and activation will probably provide new clues to guide cancer treatment [29, 53] . In general, Src is activated via autophosphorylation of the tyrosine residue Y416 in its SH1 kinase domain and inactivated by phosphorylation of the negative regulator Y527 at its carboxy-terminal domain by Csk [49, 54] . Our current results revealed that the enhanced expression of P-gp reduces cisplatininduced active pSrc Y416 levels but significantly increases inactive pSrc Y527 levels, implying that P-gp may increase the access of cytosolic Csk to membrane-anchored Src. However, overexpressed P-gp does not change Csk protein levels. Csk, existing in the cytoplasm for lacking a fatty acetylation site , must be recruited to the plasma membrane to exert its function. We further found that Cbp, and -shCbp (C) cells were treated with cisplatin at various concentrations for 1 h, washed, and incubated with fresh medium for 2 h. The levels of total Src, pSrc Y416 , and pSrc Y527 were determined by western blotting. β-actin was included as a loading control. a Csk binding protein that efficiently recruits cytosolic Csk to the plasma membrane was significantly increased in P-gp overexpressing cells. Therefore, we infer that overexpression of P-gp and Cbp may facilitate recruitment of Csk to the membrane and result in phosphorylation of Src at Y527 upon treatment with DNA-damaging agents. Src is mainly anchored to membranes via its N-terminal myristolyation and adaptor protein [18, 36] . Numerous studies have shown that P-gp and Src are associated with lipid rafts [40, 56] , where lipid raft modulation inhibits P-gp and limits the transforming potential of Src [57, 58] . Because we observed partial colocalization of P-gp, Cbp and Src on the plasma membrane, P-gp may upregulate Cbp to interfere with Src activation in cells treated with DNA-damaging agents. In addition to recruiting Csk to lipid raft-associated Src kinase, Cbp can also potentiate Csk enzymatic activity . Numerous reports have shown that the Cbp-Csk-Src pathway is involved in the regulation of Src signaling in response to diverse stimuli . Our results indicated that Cbp was closely associated with P-gp overexpression in either acquired MDR cells, KBvin10, or ectopically expressed cells, Paca-S1-P1. We may further infer that P-gp is involved in facilitating the negative regulatory Cbp-Csk-Src pathway, hence sensitizing MDR cells to certain DNA-damaging agents, such as cisplatin. The mechanism of Cbp upregulation, particularly how P-gp enhances Cbp expression, remains elusive. P-gp is a lipid translocase that induces the translocation of sphingomyelin and glucosylceramide across the plasma membrane . Recent studies have shown that P-gp interferes with membrane lipid organization and facilitates the recruitment of Cbp . Our present study demonstrated that enhanced Cbp in P-gp overexpression cells is likely because of increased transcription and protein stability. However, the interaction domains between P-gp and Cbp warrant further investigation. Our study supports our previous finding that P-gp may play a special role in the inhibition of Src activation in cells exposed to DNA-damaging agents . We further identified a critical role of Cbp in regulating P-gp-mediated Src activation and in sensitizing cancer cells to ICL agents. We also demonstrated that enhanced expression of Cbp could sensitize the xenografts to cisplatin in mice. These findings suggest that P-gp overexpressing cells might interfere with Cbp-Csk-Src cascade and DDR and consequently enhance the sensitivity to DNA damaging agents, such as cisplatin or other DNA ICL agents. Therefore, DNA ICL agents might be potential therapeutic drugs against P-gp-overexpressing MDR cells, and Cbp mimics could serve as a potential agent for anticancer therapy.
MATERIALS AND METHODS
Chemicals and reagents Vincristine, vinblastine, etoposide, paclitaxel (Taxol), cisplatin, carboplatin and melphalan were purchased from Sigma-Aldrich (St Louis, MO, USA), and doxorubicin from MP Biomedicals (Santa Ana, CA, USA). Cell culture chemicals were obtained from Gibco (Grand Island, NY, USA), and other reagents from Merck (Darmstadt, Germany). BO-1922 was synthesized according to our published procedure . Cisplatin was freshly prepared by dissolving in dimethyl sulfoxide, immediately diluted with medium, and administrated into the culture dishes or wells.
Cell lines and cell culture KB and KBvin10 cells, obtained from Dr. J. Y. Chang (National Health Research Institutes, Taiwan), were cultured as previously described . KBvin10 cells overexpressing P-gp and vincristine resistance were derived from KB cells, which were originally thought to be an oral epidermal carcinoma cell line but with HeLa cell characteristics. Paca-S1 cells, derived from a pancreatic carcinoma Mia-Paca-2 xenograft by Dr. H. C. Wu (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan), were cultured in DMEM supplemented with 10% FBS and antibiotics.
Cytotoxicity assay The cytotoxicity of various cell lines to various compounds was assessed using Presto Blue (Life Technologies Co.) according to the manufacturer's instructions. Briefly, 3,000-5,000 cells were seeded in each well of 96-well plates and incubated at 37C overnight prior to drug treatment. In general, the cells were treated with various concentrations of drugs for 72 h. At the end of treatment, an aliquot of Presto-Blue solution (at 1/10 volume) was added to each well, and the plates were further incubated at 37C for 3 h. Cell proliferation was assessed by determining the absorbance at 570/600 nm using a microplate spectrophotometer, and the IC 50 values of each compound were calculated from the dose-effect relationships at six or seven concentrations using CompuSyn software (version 1.0.1; CompuSyn, Inc.) by Chou and Martin based on the median-effect principle and plot .
Western blot analysis Western blotting was performed as previously described . Equal amounts of cell lysates or immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blotting using the following antibodies. Primary antibodies against P-gp, Csk and FANCD2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Src, pSrc Y416 , pSrc Y527 , ATM, pATM S1981 , Chk2, pChk2 T68 , Brca1, pBrac1 S1524 , Nbs1, pNbs1 S343 , Mre11 and Rad50 from Cell Signaling Technology (Danvers, MA, USA), Rad51 from GeneTex (Irvine, CA, USA), γH2AX from Calbiochem (San Diego, CA, USA), and Cbp and β-actin from Abcam (Cambridge, MA, USA). The secondary horseradish peroxidase-conjugated antibody was purchased from Abcam (Cambridge, MA, USA). The specific protein bands were visualized by chemiluminescence using the SuperSignal West Pico chemiluminescence reagent (Pierce, Rockford, IL).
Ectopic expression P-gp and Cbp were inserted into the pCMV6-ENTRY and pCMV6-AC-GFP (OriGene Technologies, Rockville, MD, USA) expression vectors, respectively. The constructed plasmids were transfected into cells by Lipofectamine 2000 according to the manufacturer's instructions. Stable transfectants were selected by culturing the cells with 0.5 g/ml G418 (Gibco) for 2 weeks. Paca-S1 cells stably expressing P-gp were maintained in the presence of 10 nM vincristine.
Gene silencing by shRNA The lentiviruses containing the expression vectors of shRNAs of P-gp (pLKO.1-shP-gp) and Cbp (pLKO.1-shCbp) were obtained from the National RNAi Core Facility located at Academia Sinica, Taipei, Taiwan. The sequences of the P-gp and Cbp shRNAs were 5′-GCTCATCGTTTGTCTACAGTT-3′ and 5′-TGATCTCTATGCTACTGTTAA-3′, respectively. The lentiviral expression vector pLKO.1-shLuc, containing 5′-GCGGTTGCCAAGAGGTTCCAT-3′ specific for luciferase, was used as a vector control. To silence the expression of P-gp or Cbp, KBvin10 cells were infected with lentivirus containing the specific shRNA. These cell lines were maintained in the presence of 4 μg/ml puromycin.
Reverse transcription polymerase chain reaction (PCR) Total RNA was extracted by using Trireagent (Sigma). The total RNAs were subjected to reverse transcription with SuperScript III (Invitrogen) for RNA synthesis. PCR was performed with the following pairs of specific primers: 5′-TCCGGTTTGGAGCCTACTTGG-3′ and 5′-AGGCATGTATGTTGGCCTCC-3′ for P-gp, 5′-TGGGGACATTCTTTCAGAGG-3′ and 5′-GGTGGAC TCCGGAACTGTAA-3′ for Cbp, 5′-CGAGATCCCTCC AAAATCAA-3′ and 5′-TGCTGTAGCCAAATTCGTTG-3′ for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). The PCR products were examined by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized under UV light.
Immunoprecipitation Cell were lysed in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8, 1% NP-40, and 0.25% deoxycholate containing a protease inhibitor cocktail set (Calbiochem) and 1 mM each of phenylmethylsulfonyl fluoride and sodium orthovanadate) and pre-cleared by incubating with protein G-Sepharose beads for 30 min. Pre-cleared lysates were incubated with the indicated primary antibody or isotype-specific IgG as a control overnight at 4C, followed by incubation with protein G-Sepharose beads (Millipore, Billerica, MA, USA) for 2 h at 4C. Immunocomplex beads were washed 3 times with PBS, immediately boiled in 2× sample buffer and then processed for electrophoresis and western blotting.
Immunofluorescence staining To visualize the intracellular location of P-gp, Src or Cbp, KBvin10 or Paca-S1 cells were cultured on slides, washed with cold PBS and fixed with 100% icecold methanol for 30 min. Slides were then washed twice with PBS, permeabilized with PBS containing 0.2% Triton X-100 for 10 min and incubated overnight with appropriate antibody at 4C, followed by Alexa Fluor 488-conjugated secondary goat anti rabbit IgG antibodies or Alexa Fluor 555-conjugated secondary goat anti mouse IgG antibodies (Molecular Probes, Eugene, OR, USA). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) as previously described . After mounting with 50% glycerol in PBS, images were acquired with a laser scanning confocal microscope (LSM 700, Carl Zeiss Microlmaging Inc., Thornwood, NY, USA) and AxioVision software. a IC 50 values are the drug concentrations that inhibit cell growth by 50%. The data are expressed as means ± SE of three independent experiments. b RF, resistant factor (IC 50 of Paca-S1-P1 cells/IC 50 of Paca-S1-V cells).
Figure 1 : 1 Figure 1: Increased susceptibility to DNA damaging agents and decreased Src activation in P-gp overexpressed KBvin10 cells. (A) Increasing cytotoxic effects of DNA damaging agents to KBvin10 cells. KB and KBvin10 cells were treated with various concentrations of cisplatin or BO-1922 for 1 h, washed with phosphate buffered saline (PBS) and then incubated with fresh medium for 72 h. The cell proliferation was analyzed using Presto-Blue. Bars are SD of three independent experiments. (B) No significant Src activation in KBvin10 cells treated with cisplatin. KB or KBvin10 cells were treated with various concentrations of cisplatin for 1 h, washed with PBS, and incubated with fresh medium for 2 h. The levels of Src, pSrc Y416 (active) and pSrc Y527 (inactive), EGFR and pEGFR Y845 were determined by western blotting. β-actin was included as a loading control.
Figure 2 : 2 Figure 2: Attenuation of cisplatin-induced Src activation in P-gp overexpressing Paca-S1 cells. (A) Enhanced expression
Figure 3 : 3 Figure 3: Reduced DNA damage response in cisplatin-treated P-gp overexpressing cells. (A) Accumulated gH2AX in Paca-S1-P1 cells treated with cisplatin. Paca-S1-V and Paca-S1-P1 cells were treated with 50 μM cisplatin for 1 h, washed and cultured in drug-free medium for various time periods. The protein levels of γH2AX were determined by western blotting. (B) Attenuation of DNA repair protein activation by P-gp. Paca-S1-V and Paca-S1-P1 cells were treated with 50 μM cisplatin and KB and KBvin10 cells with 20 μM cisplatin for 1 h, washed, and incubated for 0, 1, 3 and 6 h. Afterward, several DDR related proteins with or without activated phosphorylation, such as ATM and pATM S1981 , Chk2 and pChk2 T68 , Brca1 and pBrca1 S1524 , Nbs1 and pNbs1 S343 , and total proteins of Mre11, Rad50, Rad51, and FANCD2 were analyzed by western blotting. β-actin was included as a loading control.
Figure 4 : 4 Figure 4: Increased Cbp expression and stability in P-gp overexpressing cells. (A and B) Increased Cbp protein (A) and mRNA (B) in P-gp-overexpressing cells. The protein levels of P-gp, Cbp and Csk and the mRNA levels of P-gp and Cbp in KB, KBvin10, Paca-S1-V and Paca-S1-P1 cells were analyzed by western blotting and RT-PCR, respectivley. β-actin was included as a loading control in western blotting assay, while GAPDH expression served as an internal control of RT-PCR assay. (C) Increased Cbp stability in P-gpoverexpressing KBvin10 cells. KB and KBvin10 cells were incubated with cycloheximide (CHX) (20 μg/ml) for the indicated time periods. The protein levels of Cbp were determined by western blotting (left). β-actin was included as a loading control. The relative intensity of Cbp (right) was determined by imaging software. Bars are SD of three independent experiments.
Figure 5 : 5 Figure 5: Interaction among P-gp, Src, and Cbp. (A) Co-immunoprecipitation of P-gp, Src, and Cbp. Cell lysates from KB and KBvin10 cells were immunoprecipitated with antibodies against P-gp, Src or Cbp. The immuno-complexes were reciprocally subjected to western blotting using P-gp, Src, and Cbp antibodies, respectively. (B) Co-localization of P-gp, Src, and Cbp in KBvin10 cells. Logarithmically growing cells were fixed and stained with immunofluorescent antibodies as described in MATERIALS AND METHODS. The images were acquired by confocal microscopy.
Figure 7 : 7 Figure 7: Increased resistance to DNA-damaging agents by P-gp or Cbp silencing. (A) Reduced P-gp or Cbp by shP-gp or shCbp, respectively. KBvin10 cells were infected with lentiviral shLuc, shP-gp, or shCbp for 24 h, washed, and cultured in medium containing 4 μg/ml puromycin. Four days after selection, P-gp and Cbp proteins in KBvin10-shLuc, -shP-gp, or -shCbp cells were analyzed by western blotting. β-actin was included as a loading control. (B) Increased survival rate in P-gp-or Cbp-silenced cells treated with DNA-damaging agents. KBvin10-shLuc, -shP-gp, or -shCbp cells were treated with various concentrations of cisplatin for 1 h, washed with PBS, and incubated for 72 h. The cell proliferation was determined using Presto-Blue agents as described previously. Bars are SD of three independent experiments. (C) Decreased accumulation of gH2AX in cisplatin-treated KBvin10-shCbp cells. KBvin10-shLuc and -shCbp cells were treated with 10 μM cisplatin for 1 h, washed, and changed to drug-free medium for the indicated time. γH2AX levels were analyzed by western blotting. (D) Facilitating the repair of cisplatin-induced DNA ICLs in Cbp silencing cells. KBvin10-shLuc and KBvin10-shCbp cells were treated with 10 μM cisplatin for 1 h. Cisplatin-induced DNA ICLs were determined by a modified comet assay after incubation in drug-free medium for different time periods. Data are average of 2 independent experiments.
Figure 8 : 8 Figure 8: Enhanced Src activation in P-gp-or Cbp-silenced KBvin10 cells by cisplatin. KBvin10-shLuc (A), -shP-pg (B),
Figure 9 : 9 Figure 9: Attenuated Src activation by cisplatin in Cbp-overexpressing cells. (A) Enhanced Cbp expression in KB cells transfected with the Cbp expression vector. Stable cell lines were cloned and maintained in medium containing G418. The protein levels of Cbp in KB-AcGFP cells and KB-AcGFP-Cbp cells were analyzed by western blotting. (B) Increased sensitivity to DNA-damaging agents in Cbp-overexpressing cells. KB, KB-AcGFP and KB-AcGFP-Cbp cells were treated with cisplatin for 1 h, washed and then incubated with fresh medium for 72 h. The cell proliferation was determined using Presto-Blue. (C) Attenuation of Src activation by Cbp. KB-AcGFP and KB-AcGFP-Cbp cells were treated with cisplatin at various concentrations for 1 h, washed, and incubated with fresh medium for 2 h. The levels of total Src, pSrc Y416 , and pSrc Y527 were determined by western blotting. β-actin was included as a loading control. (D and E) Increased anti-tumor activity of cisplatin by enhanced Cbp expression. Athymic nude mice bearing cancer xenografts, derived from KBvin10-shLuc and KBvin10-shCbp cells (D) and from KB-GFP and KB-Cbp cells (E) were intratumorally treated with one dose of saline or cisplatin (4 mg/kg body weight) when the tumor reached a volume of 100 mm 3 (6 mice/each group). Ten days after treatment, the tumor volumes were determined. Each bar represents the mean ± S.E of 6 mice from 2 independent experiments. Statistical significance was determined by one-way ANOVAs. *p < 0.05, saline vs. cisplatin; # p < 0.05, KBvin10-shLuc vs KBvin10-shCbp or KB-GFP vs KB-Cbp cells treated with cisplatin.
Table 1 : The IC 50 values of various drugs against Paca-S1-V and P-gp-overexpressing Paca-S1-P1 cells a Drugs Paca-S1-V Paca-S1-P1 RF b 1 Vincristine (nM) 3.4 ± 1.2 37,270 ± 317 10,961 Vinblastine (nM) 1.6 ± 0.0 317.2 ± 4.5 198 Doxorubicin (nM) 20.2 ± 6.5 1,389 ± 30.1 69 Etoposide (μM) 2.9 ± 0.3 35.4 ± 4.3 12 Paclitaxel (μM) 0.03 ± 0.00 2.0 ± 0.2 67 Cisplatin (μM) 19.6 ± 0.4 12.6 ± 2.8 0.64 Carboplatin (μM) 56.1 ± 1.8 51.4 ± 0.5 0.91 Melphalan (μM) 109.6 ± 4.0 66.5 ± 5.4 0.61 BO-1922 (μM) 0.52 ± 0.02 0.26 ± 0.08 0.50
www.impactjournals.com/oncotarget
|
10.1155/2012/840247
|
cc-by
| null | null |
openalex
|
Chronic treatment with levodopa (LD) in Parkinson's disease (PD) can cause drug induced dyskinesias. Mucuna pruriens endocarp powder (MPEP) contains several compounds including natural LD and has been reported to not cause drug-induced dyskinesias. We evaluated the effects of Mucuna pruriens to determine if its underlying mechanistic actions are exclusively due to LD. We first compared MPEP with and without carbidopa (CD), and LD+CD in hemiparkinsonian (HP) monkeys. Each treatment ameliorated parkinsonism. We then compared the neuronal firing properties of the substantia nigra reticulata (SNR) and subthalamic nucleus (STN) in HP monkeys with MPEP+CD and LD+CD to evaluate basal ganglia circuitry alterations. Both treatments decreased SNR firing rate compared to HP state. However, LD+CD treatments significantly increased SNR bursting firing patterns that were not seen with MPEP+CD treatments. No significant changes were seen in STN firing properties. We then evaluated the effects of a water extract of MPEP. Oral MPWE ameliorated parkinsonism without causing drug-induced dyskinesias. The distinctive neurophysiological findings in the basal ganglia and the ability to ameliorate parkinsonism without causing dyskinesias strongly suggest that Mucuna pruriens acts through a novel mechanism that is different from that of LD.Introduction Dopamine replacement therapy with LD+DDCI (dopa decarboxylase inhibitor) is the most effective pharmacological treatment for PD. However, LD+DDCI remains expensive and out of reach of many PD patients in developing countries and causes disabling drug-induced dyskinesias, motor fluctuations, and neuropsychiatric complications in most PD patients . Development of an oral treatment with the same or higher efficacy of LD+DDCI that does not cause drug-induced complications is an unmet need. MPEP in Ayurvedic medicine provides alleviation of parkinsonism but has been reported to not cause druginduced dyskinesias . MPEP contains 4-5% natural LD, which had been implicated as its main mechanism of action and the reason for not causing drug-induced dyskinesias (i.e., did not contain enough LD). Despite MPEP being well-tolerated in clinical trials , PD patients complain of inability to consume a large volume (30 g) of this leguminous protein as it often causes adverse gastrointestinal side effects. Therefore, despite wide availability of MPEP as a nutraceutical via Internet marketing and in Ayurvedic pharmacies, MPEP is rarely utilized as allopathic treatment for PD even in India [1, 11] . This lack of popularity of MPEP is related to its gastrointestinal side effects and the erroneous notion that MPEP simply represents a natural form of lowdose LD. However, MPEP is ubiquitously used by Ayurvedic physicians worldwide as an ingredient in medications for the treatment of PD.
Evidence-Based Complementary and Alternative Medicine We recently reported that a newly formulated, simple MPEP water extract (MPWE) given parenterally significantly ameliorates parkinsonism with reduced drug-induced dyskinesias in the HP rat . This suggested that its anti-PD and antidyskinetic effects could not be explained by the presence of small quantities of natural LD alone and that MPWE is more effective when used alone without the addition of a DDCI. These surprising findings led us to further investigate the unique anti-PD and anti-dyskinetic properties of MPEP and MPWE in the 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-(MPTP-) treated parkinsonian monkey. Further evaluation of the mechanistic actions of MPEP and MPWE may increase its potential as an alternative therapy for PD.
Materials and Methods 2.1. Animals. Fourteen adult (6-9 kg) rhesus (Macaca mulatta) and two cynomolgus (Macaca fascicularis) monkeys received either intracarotid (ICA) MPTP to induce an HP state, ICA+systemic (IV) MPTP to induce an overlesioned HP (OHP) model, or systemic (IM or IV) injections of MPTP to induce bilateral parkinsonism . Each animal was operant-conditioned behaviorally trained to accept medications to ensure proper consumption and clinical oral simulation. Clinical assessments were taken after MPTP to ensure stability of parkinsonism and at subsequent treatment exposure using the modified version of the Unified Parkinson's Disease Rating scale for primates (mUPDRS) . Electrophysiological recordings before and after treatments were done in awake, behaving parkinsonian animals. Standard extracellular single-cell recording techniques were used as described in detail elsewhere . All procedures were in compliance with the "Principles of Laboratory Animal Care" (NIH no. 86-23, revised 1985) and approved by the institutional animal care and use committee.
Administration of MPTP to Induce Parkinsonism. For ICA administration of MPTP to create an HP state, animals were placed under deep general anesthesia, the left common carotid artery was exposed, and the internal carotid artery was isolated followed by manual retrograde injection of MPTP solution (0.5 mg/kg body weight at a concentration of 1 mg/mL) over a period of 15 minutes. The animal was allowed to recover and assessed for stability of HP. Depending on stability of HP state (see below for behavioral testing details), exposure to ICA MPTP was performed up to 4 times in each animal. Repeat surgeries were not performed before 2 weeks of observation had been completed and surgical scar from the previous surgery had healed. The cumulative ICA MPTP dose ranged from 0.5 to 2.5 mg/kg. A subset of animals was rendered overlesioned HP (OHP). To achieve an OHP state, animals were initially treated with ICA MPTP. Once HP state was stable, the animal received subsequent injections of IV MPTP (0.2 mg/kg), inducing mild parkinsonism in the previously unaffected side. Another set of animals was rendered bilaterally parkinsonian with systemic IV or IM injections of MPTP (0.2 mg/kg). Cumulative doses of systemic MPTP ranged from 0.2 mg/kg to 1.0 mg/kg. Drug treatments were then given only when animals were stable parkinsonian for >3 months as determined by no changes in the mUPDRS ratings (see below) performed twice each month separated by a minimum of 15 days and operantconditioned for a minimum of 6 months such that they were compliant with oral dosing of medications (see the section below).
Operant Conditioning for Oral Medication Compliance without Compromising Enrichment Protocols in Parkinsonian Primates. The following protocol was used for operant conditioning and behavioral training in each animal to ensure compliance and complete consumption of antiparkinsonian medications. Each animal was individually housed such that visual and olfactory contact with conspecifics was maintained at all times. Various types of toys were placed in each cage and rotated every other week. At any one time, every cage contained a hanging toy, such as a ball or a mirror and at least one (usually two) chewing toy, such as a Hercules dental chew toy, Dental ball, Kong toys, nylabones, or pieces of wood (Bio-Serv). In some instances, certain animals showed adverse stress reactions to certain types of toys. In this case, that toy was removed from the animal's cage and replaced with another toy. In addition to the regular feed, monkey diet was supplemented with fruits, vegetables, nuts, or other types of "treats" every day. The size, quantity, and time of day that these "treats" were given (always in the late afternoon after training) were monitored carefully so as not to interfere with our behavioral training. Also, each day, Monday-Friday, the animals were presented with an enrichment activity. These activities included watching cartoons, "novel" food day, foraging devices, and special activities such as air-popped popcorn or bowls of water to play with. On weekends, the animals were given extra food treats. Cages were checked for any remaining monkey biscuits or treats each evening; any remaining food was removed in order to maintain the appropriate level of food scheduling necessary for operant training of a food-picking task or voluntary consumption of medications. All animals were observed for stereotypical (pacing, rocking, digit-sucking) and self-injurious behaviors (self-biting, head banging). If any such behaviors were seen, enrichment for those animals was increased. Oral LD and MPEP were successfully administered twice daily (AM and PM) by hiding powdered drug in food treats. MPWE was typically given orally without the need of hiding it in any additional foods or liquids. Animals appeared to enjoy MPWE and voluntarily consumed it completely at each dose. Improvement in parkinsonism was then assessed by using the mUPDRS and confirmed through analysis of blood plasma levels in representative animals at the same time as the mUPDRS exams. Animals were continuously monitored to ensure complete consumption and drug compliance during treatments by investigators. Depending on the desired task (consumption of treatment or interaction with investigator to evaluate mUPDRS), any of the enrichment protocols in Table 1 could be detrimental to operant conditioning. Similarly, any of the training conditions could cause behavioral problems in monkeys due to lack of appropriate enrichment. To overcome these barriers to operant conditioning, we limited the time when enrichment was given (all enrichment was given after training session to maintain food scheduling), scheduled enrichment only in the afternoon or following the end of a testing session, and maintained visual, sound, and olfactory stimuli without interfering with operant conditioning. The same individuals were involved in the enrichment, operant conditioning, and husbandry to limit distress and to reinforce the operant conditioning. Veterinary care interaction with these monkeys with individuals other than the persons involved in operant conditioning and enrichment was limited to yearly physicals and twice a year TB testing and any other USDA mandated veterinary checkups.
Clinical Assessment. Behavioral ratings were performed using the mUPDRS in a blinded fashion. A more detailed description can be found in our previous publications [15, 16] . Briefly, the mUPDRS consists of subjectiverater-dependent but validated and reliable blinded evaluations of vocalization/hooting, facial expression, tremor (rest or action), muscle tone/rigidity, hypokinesia, finger dexterity, foot agility, balance/postural instability, spontaneous gait, dystonia, and circling/dyskinesia. Each item on the mUPDRS has a range from 0 (no motor deficits) to 4 (very severe impairment) for each limb or body part and is modeled after the UPDRS was used to rate PD patients in clinical trials. Animals were also further assessed for druginduced dyskinesias using a modified Abnormal Involuntary Movements Scale (AIMS) previously described for primates [18, 19] . AIMS scores are represented as the total sum of dystonic posturing and choreiform movements in the face, trunk, and each limb. Severity was evaluated using the following scale: 0 = none; occasional, mild = 1; intermittent, moderate = 2; continuous, severe = 3. The entire clinical rating session was videotaped for minimum of 4 hours after each dose of medication for a minimum of 8 hours of video in representative animals. mUPDRS and AIMS scores were taken at stable parkinsonian baseline state, placebo, and at an average time of 75 minutes after drug treatments. We established the optimal LD/CD dose for each animal using blinded testing every 2 weeks at monthly intervals starting at a dose of 50 mg LD/12.5 mg CD b.i.d. (i.e., LD 100 mg/CD 25 mg/day) and escalated by 100 mg LD every 2 weeks to achieve no further improvements in mUPDRS scores despite dose escalation. Thereafter, the lowest dose of LD that produced the largest improvement in mUPDRS scores was chosen as the optimal dose of LD in each animal. Thus, animals were tested on LD treatment for a minimum of 3-4 months to determine their optimal LD dose. All animals were washed out of LD treatments for 1 month before initiating MPEP or MPWE treatments. In a similar fashion, optimal dose of MPEP, and MPWE was also determined and optimal behavioral plateau that coincided for LD, LD+DDCI, MPEP, and MPWE was identified. This plateau phase was used for all the experiments described in this paper to make comparisons with equipoise and validity. 2.1.4. Electrophysiology. After recordings were taken during placebo and on drug treatments, the recordings were sorted by offline principal component analysis, and interspike intervals (ISIs) were generated. Acceptable records were comprised of at least 400 spikes and had duration between 60 and 120 sec. Firing rates and seven measures of the firing patterns were employed: the coefficient of variation (CV) of the ISIs, the burst index (mean of the ISI distribution divided by the mode ), the percent of spikes in bursts and percentage of time in bursts calculated by the Poisson surprise method , the density discharge histogram (DDH) compared to the DDH of a random Poisson spike train , the range of the DDH, and the sample entropy . The seven numeric firing pattern metrics were compared using the Wilcoxon-Mann-Whitney rank-sum test, and the categorical DDH classification was compared using Fisher's 2 × 2 exact test (grouping together "Poisson" and "bursty" categories).
Drug Treatments
Preliminary Dose-Finding and Toxicity Studies of MPEP: Experimental Design and Results. We first completed a preliminary dose-finding study to determine optimal doses and toxicity adverse effects of MPEP and MPEP+CD. HP monkeys were tested on MPEP and MPEP+CD (25 mg) to find optimal dosing after attaining stable HP state (N = 3). Each treatment epoch was followed by 2 weeks of washout. Two separate mUPDRS scores 14 days apart were obtained on placebo and for each treatment epoch. MPEP was titrated in these studies from 6 g/day to the highest dose of 18 g/day (N = 11) to evaluate gastrointestinal effects, druginduced dyskinesias, or behavioral correlates of psychiatric symptoms. Blood draw was performed 90 minutes after administration and consumption of medications (placebo, LD+CD (250 mg/62.5 mg) and MPEP+CD (4.5 g/25 mg)) to test the bioavailability of orally administered LD and MPEP at approximately equivalent doses for pharmacological estimation of dopamine levels. See Table 2 for representative drug-dosing block design. In these initial studies, mean mUPDRS scores improved by 4% (change from 35 to 33.5), 24.2% (35 to 26.5), and 27.1% (35 to 25.5), respectively, with 3 g, 6 g, and 9 g (total daily dose) of MPEP alone. Optimal dose for MPEP+CD was determined to be 9 g of MPEP + 50 mg of CD/day. Table 2 : Dosing regimen for non-human primates. A block design was devised for each testing session. Each behavioral testing cohort was varied to maintain blind and to prevent the behavioral rater from guessing the treatments. Each such block design was repeated twice for each experiment and videos are independently rated. Representative animals were videotaped continuously in the room 24 × 5 × 365 days. The postdrug treatment videos were culled from these videos by the person who administered the drug who was unblinded. These culled video segments were used for the scoring along with the rater executed direct observational scoring of mUPDRS and AIMS. These culled segments began as soon as the person administering the drug confirmed successful consumption of the drug and lasted 8 hours from that time. The notion of the average time of 75 minutes refers to the average time at which behavioral ratings using mUPDRS and AIMS were scored for the study. The validity of this time frame for the detection of optimal effects of LD and LD/DDCI oral treatments has been published previously. To make meaningful valid comparisons MPEP and MPWE treatments were also performed at the same time schedule. The remainder of the video was rated, but it does not have the observer interaction and it only shows routine animal activity in its home cage. As expected, LD and LD/DDCI treatments had behavioral benefits that lasted 180 minutes and then ameliorated. Effects of MPEP and MPWE lasted longer and appeared to dissipate only after 6 hours.
Experiment 1: Effects of MPEP, MPEP+CD, and LD+CD. After finding preliminary optimal doses of MPEP, we examined the effects of 4.5 g MPEP alone (∼225 mg LD) (N = 5), 4.5 g MPEP (∼225 mg LD) + 25 mg CD (N = 6) and 100-200 mg LD + 25-50 mg CD (i.e., daily doses were 9 g MPEP alone (N = 5), 9 g MPEP+50 mg CD (N = 6), and 200-400 mg levodopa + 50-100 mg CD (N = 6)) in HP and OHP primates. In a subset of animals, cranial recording chambers were surgically implanted to permit chronic singlecell extracellular neuronal recording from the left STN and SNR in the stable HP state (N = 3), with LD+CD treatment (N = 2) and with MPEP+CD treatment (N = 1) (a portion of this study has been presented elsewhere ). HP, OHP, and bilateral parkinsonian animals (N = 5) were treated with placebo, LD+CD or MPWE b.i.d. for 3-11 days. mUPDRS scores were obtained on placebo, oral LD (1 to 3.5 tablets LD/CD-100/25), and escalating doses of 4 mL-36 mL MPWE orally (∼96 mg-864 mg LD, resp.) until optimal doses were found using a blinded, randomized design with 2 weeks of washout between treatments. When compliance was an issue with oral LD+CD, animals received systemic injections of LD at the equivalent optimal doses of oral LD (LD methyl ester with benserazide (BZ)). Drug-induced dyskinesias were assessed using the abnormal involuntary movements (AIMS) rating scale in animals that displayed clear LD-induced dyskinesias similar in advanced PD [15, 18, 19] . Data was analyzed using ANOVA with Tukey posttest or Chi-square test (mean ± SEM).
Results
MPEP and LD Effects on Parkinsonism and Basal Ganglia Electrophysiology. mUPDRS scores on placebo were 15.6 ± 2.6 and decreased to 8.0 ± 1.6 with MPEP alone, 7.7 ± 1.5 with MPEP+CD, and 4.5 ± 1.1 with LD+CD, optimal doses (Figure 1 ). These doses caused no observable adverse effects. A portion of this electrophysiological data has been presented in a previously published report . SNR firing rate showed significant reduction in SNR on both LD+CD and MPEP+CD. STN firing rate showed no significant difference, but a trend toward reduction on MPEP+CD (Figure 2(a) ). SNR firing pattern became more bursty on LD+CD, measured by Poisson DDH comparison. SNR patterns changes on MPEP+CD did not reach statistical significance, but showed a trend toward increased burstiness, but not as pronounced as LD+CD. STN patterns did not show a statistical change, although both LD+CD and MPEP+CD showed a trend toward reduction of the number of bursty neurons (Figure 2(b) ). Median SNR normalized coefficient of variation was higher on LD+CD than baseline HP state. On MPEP, the SNR showed a trend toward increased normalized CV, but it was not significant. There were no significant changes in the STN (Figure 2 not show any statistically significant changes. However, there was a trend for both LD+CD and MPEP+CD to make the SNR more bursty, with MPEP+CD showing a smaller effect than LD+CD. There was a trend for LD+CD to make the STN less bursty, and MPEP+CD showed the same trend to an even larger degree (Figures 2(d ) and 2(e)). DDH range counts were not statistically different in the different conditions. However, there was a trend for LD+CD to make the SNR more bursty. MPEP+CD showed a trend toward making the SNR more bursty but it was not significant (Figure 2 (f)). The burst index was not statistically significant between groups. However, there was a trend for LD+CD to make the SNR more bursty, which was not replicated on MPEP+CD. In fact, there was a trend for MPEP+CD to reduce the burstiness of SNR. In the STN, the trends were reversed, but again neither were significant (Figure 2 (g)). Sample entropy did not show any significant differences, although the MPEP+CD treatment slightly reduced the SNR sample entropy and slightly increased the STN sample entropy (Figure 2 (h)).
MPWE Ameliorates Parkinsonism without Causing Drug- Induced Dyskinesias. mUPDRS scores on placebo were 18.0 ± 5.6, which significantly decreased with optimal doses of MPWE treatments (5.4 ± 0.4) and LD+CD treatments (5.3 ± 1.9) (Figure 3 (a)). Average optimal dose of LD was 250 mg and optimal dose of MPWE was 20 mL (∼480 mg LD) b.i.d. in this experiment that included HP, OHP, and bilateral parkinsonian animals. MPWE caused no apparent GI problems or drug-induced hallucinations. However, LD+CD treatments produced significant druginduced dyskinesias (AIMS score = 7.3 ± 1.3) in two bilaterally parkinsonian animals, whereas no apparent druginduced dyskinesias were observed with MPWE treatments (AIMS score = 0) (Figure 3(b) ).
Discussion In the present study, we demonstrate that Mucuna pruriens in powder and water extract form can significantly ameliorate behavioral deficits in primate models of PD. We also demonstrate that the mechanistic actions of Mucuna pruriens cannot be attributed to LD alone and that Mucuna pruriens has a unique mechanism of action on the basal ganglia electrophysiology that is different from that of LD when tested at equivalent doses. This is a confirmation of earlier suggestions that the anti-PD effects of Mucuna pruriens were not simply due to natural LD. Indigenous medicines based on natural products like Mucuna pruriens are often unique in that they contain several constituents in combination. Mucuna pruriens has over 50 known constituents that have been identified to date (Table 3 ) and perhaps others yet to be discovered . Identifying the single component or combination of components in Mucuna pruriens responsible for its anti-parkinsonian/antidyskinetic effects is daunting. Although identification of each individual component and its exact quantity required to reproduce these effects is theoretically possible, such a task is time consuming and expensive. Mucuna pruriens is widely farmed in many countries as an intercrop and is exceedingly inexpensive to produce as a standardized natural product with uniform efficacy. Thus, this renewable, natural product may represent a new treatment that is different from contemporary drug discovery methods where identification of active ingredients, synthetic manufacture, safety, and efficacy testing followed by mass marketing of the synthesized compounds is replaced by a strategy that focuses on identification of safety and efficacy of a standardized natural product and its mechanism of action when used as a whole. While such an approach may sound counterintuitive, archaic, and confrontational to the current wisdom of scientific advancement, it is pragmatic and has the potential to advance the therapy of PD with the possibility of worldwide availability of inexpensive Mucuna pruriens formulations.
Effects of Mucuna pruriens Endocarp Powder. In experiment 1, we found that MPEP had to be dosed at higher quantities to get maximal effect when compared to LD (100-200 mg synthetic LD versus 225 mg natural LD in MPEP). The large volume of MPEP powder (6 g to 18 g/day) in preliminary studies and in experiment 1 was very difficult to successfully administer in monkeys due to gastrointestinal side effects, similar to those of earlier reports [1, . These gastrointestinal effects could be in part due to the large protein content in this leguminous cotyledon powder, a well-known cause of abdominal bloating, flatulence, and gastrointestinal irritability. Serum dopamine measurements demonstrate that bioavailability and peak plasma pharmacokinetics of natural LD contained in MPEP and synthetic LD are quite similar. This finding further strengthens the notion that MPEP contains additional anti-PD and antidyskinetic agents beyond the 4-5% natural LD content. Previous reports have demonstrated that LD and other dopamine replacement therapies can significantly alter firing properties of basal ganglia nuclei. This has been discussed in our recently published paper in detail [16, 20, . We demonstrate that SNr firing rate is significantly decreased after treatment with both LD and MPEP. However, LD treatment did not decrease STN firing rate. Interestingly, MPEP did cause a trend in decreasing STN firing rate. We also found differential firing patterns between the two treatments. LD caused a significant increase in SNr bursting activity but this increase was not seen with MPEP. We also found slight differences between LD and MPEP in the other measures of burstiness. Various pharmacological agents are known to alter basal ganglia firing patterns, which include serotonin, N-methyl-D-aspartate modulators, and dopamine agonists [29, ; the presence of similar compounds in MPEP may account for the differential bursting firing patterns of MPEP.
Effects of Mucuna Pruriens Endocarp Powder Water Extract. The ameliorative effects of oral MPWE treatments are similar to the anti-PD effects of LD+CD treatment, a gold standard for pharmacological therapeutic efficacy in PD, sans its deleterious side effects in the parkinsonian primate. As shown in experiment 2, MPWE provides a simple and inexpensive solution to these problems with gastrointestinal intolerance of MPEP and demonstrates that the anti-PD and anti-dyskinetic compounds contained in Mucuna pruriens are water soluble and effective without the need for concomitant DDCI. This suggests that even monkeys "primed" to develop drug-induced dyskinesias from repeated exposure to LD+CD treatments can be successfully treated with MPWE without causing dyskinesias. Furthermore, the anti-PD and anti-dyskinetic effects of MPWE were not diminished by chronic exposure, drug washout, and reexposure. We have recently shown that LD does not cause dyskinesias in HP rhesus monkeys . In this context, we demonstrate the anti-PD properties of MPWE in HP and OHP primates, models that represent restricted nigrostriatal dopaminergic loss, and the anti-PD and anti-dyskinetic effects in the bilateral parkinsonian monkey, a model that represents more severe PD and readily exhibits drug-induced dyskinesias. These findings in the MPTP-treated monkey models are similar to what we have reported in the HP rat . Some investigators have argued that drug-induced abnormal involuntary movements displayed by the parkinsonian rat are not equivalent of drug-induced dyskinesias seen in PD and believe that the phenomenology of drug-induced dyskinesias in the primate model more closely resembles clinical drug-induced dyskinesias . We address this in the current study, confirming the preclinical relevance of the anti-PD and anti-dyskinetic effects of MPWE without a DDCI in parkinsonian primates. Taken together, these studies in the rodent and primate models of PD provide compelling preclinical evidence of the efficacy and safety for MPWE. Biochemical measurements previously mentioned provide proof that LD+CD treatments were appropriately dosed in animals that developed drug-induced dyskinesias in the MPWE experiments. We hypothesize that the improved safety profile of MPWE may be due to additional beneficial compounds as speculated in previous studies [8, 25, 26] . We escalated the dose of MPWE more than what was needed to match the optimal anti-PD effects obtained from LD+CD treatments (MPWE doses up to >1600 mg LD equivalent dose per day). Nonetheless, these animals tolerated these large doses without adverse effects. MPWE contains 4-5% LD that is identical to the 4-5% LD content reported for MPEP powder. Since MPWE was administered without a DDCI, we hypothesize that the effects cannot be entirely due to LD content in MPWE because LD would be metabolized via peripheral DDC, suggesting that MPWE may have DDCI-like activities or other mechanisms to protect LD degradation via the action of peripheral DDCI. However, our previous rodent studies suggest that MPWE action cannot be accounted for just on the basis of its purported DDCIlike activity . Thus, our rodent studies and the current experiment collectively provide behavioral evidence that the anti-PD effects of MPWE cannot be explained by the presence of 4-5% natural LD alone or the combination of natural LD and a yet-to-be identified DDCI constituent. Other water-soluble compounds that remain unidentified contained in MPWE have to be implicated for the anti-PD and anti-dyskinetic effects observed in these studies. Evidence-Based Complementary and Alternative Medicine
Oral Administration of Antiparkinsonian Treatments to Parkinsonian Monkeys. This is the first report of any phytomedicine that has been tested in primates using operant conditioned methods for oral voluntary consumption to simulate clinical PD pharmacotherapy, using placebo controls and a blinded prospective study design. This study design could represent an ideal method to perform future preclinical studies of phytomedicines in PD. We found compliance with oral consumption easier with MPWE compared to MPEP, MPEP+CD, or LD+CD. Previous studies with various MPEP formulations [8-10, 39, 40] have a number of disadvantages that include variable behavioral assessments, use of concomitant medications, inadequate washout, lack of LD dose controls, and excess variability in study populations (see detailed discussion in our recent report ). In the present study, we overcame these disadvantages by (1) using well-established primate models of PD that exhibit motor fluctuations and drug-induced dyskinesias that closely resemble its phenomenology to patients with PD, (2) ensuring drug compliance to replicate the clinical experience of PD patients, (3) utilizing the same behavioral rater for all mUPDRS assessments to eliminate interrater variability, and (4) ensuring that animals received no concomitant medications.
Conclusion We demonstrate that Mucuna pruriens and MPWE have unique mechanistic properties that are differential from LD and that the unique combination of constituents within Mucuna pruriens contributes to both its anti-PD and antidyskinetic effects. This will be advantageous to PD patients who currently take LD-containing formulations and have to experience its long-term side effects that often require invasive surgical intervention. This study also shows that MPWE contains a yet-to-be investigated portfolio of anti-PD and anti-dyskinetic agents that could open up new therapeutic avenues for PD, yet constitute a daunting and expensive conventional drug discovery approach. While additional scientific studies to identify these individual anti-PD and anti-dyskinetic components contained in MPWE may be warranted, parallel studies to evaluate the clinical use of MPWE as a safe and effective alternative to LD therapy in PD is also immediately indicated with our demonstration of its unique beneficial mechanisms of action. Figure 1 : 1 Figure 1: Comparison of mUPDRS scores in parkinsonian primates with placebo, MPEP alone, MPEP+CD, and LD+CD demonstrates significant amelioration of parkinsonism after treatments. * P < 0.05, * * P < 0.01.
6 EvidenceFigure 2 : 62 Figure 2: (a) Firing rates of SNR and STN in the HP monkey in stable HP state (baseline) and on LD+CD (Levodopa) and MPEP+CD (Mucuna) (Kruskal-Wallis P < 0.01, * * P < 0.01 rank-sum using Tukey's HSD correction, compared to baseline HP state). (b) Poisson comparison of SNR and STN neurons Pre-LD (stable baseline HP state) (Fisher's 2 × 2 two-sided exact test grouping "Poisson" category together with "regular," * P = 0.0164), Post-LD (LD treatments), and Post-MP (MPEP+CD). (c) Coefficient of variation at HP state and with treatments (Kruskal-Wallis P < 0.05, * P < 0.05 rank-sum using Tukey's HSD correction, compared to baseline HP) (d-g) Measures of firing patterns in the SNR and STN in HP state and on treatments. (h) Sample entropy of SNR and STN.
Figure 3 : 3 Figure 3: (a) A water extract of MPEP (MPWE) significantly reduces parkinsonism in the parkinsonian primate at optimal doses similar to LD+CD ( * P < 0.05) (b) and does not cause dyskinesias (P = 0.045, Chi-square test).
Table 1 : 1 Conditions for operant conditioning in parkinsonian primates. Ideal conditions for training Ideal conditions for enrichment Single-housed animals Group-housed animals Supplemental toys without food Supplemental toys containing (mirrors, chew toys) food (foraging devices) No visual contact with conspecifics Visual contact with conspecifics No sound Sound (movies, radio, wildlife sounds)
Table 3 : 3 Known components of Mucuna pruriens. Arachidic acid
|
10.26794/2308-944x-2019-7-3-45-82
|
cc-by
| null | null |
openalex
|
This article discusses some issues connected with studies of the behavioural factors when making financial decisions. Therefore, it is possible to take into account factors that are inexplicable in traditional models. The main goal of our research was verification of the hypothesis market participants make financial decisions based on their experiences, intuition, stereotypes, illusions, emotions and not only on the criterion of financial gain and rational assumptions. After all, such diverse behaviour en masse influences the financial system as a whole. The practical significance of reported here study is the identification of errors in the application of classical economic theory and the possibilities of their further elimination. An effective behavioural model to avoid negative consequences is the primary tool in making financial decisions. In the first part, the author analyses the theoretical basis of her study. The second part examines the main problems associated with classical economic theory and presents the main mistakes in making financial decisions. Particular attention the author paid to the study of the behaviour of investors and managers. The third part described the research of behavioural mechanisms in making financial decisions with specific examples and implementation of the use of mathematical models.Introduction The performance of any financial and economic entity, its long-term success and a stable position on the market are primarily determined by the effectiveness of financial policies, the quality and adequacy of financial decisions. The classical economic theories assume that the process of making financial decisions is an absolutely rational one. However, classical economic theories do not take into account the fact that there are humans at the centre of any financial processes. Thus, the main problem is that economists use models that replace the notion of "ordinary person" with "rational person". In contrast to the ideal "rational person", "ordinary people" make mistakes and take wrong decisions. Therefore, economic models and theories give erroneous forecasts and lead to negative consequences. The general assumption made under the particular research question is that people making decisions, including top management, do not always act rationally. In most cases, people make decisions based on experience or intuition. The classical theory does not take into account these factors. Thus, making such decisions is inefficient and does not lead to the desired financial results. We should also take into consideration the possibility of irrational behaviour. Therefore, in our research, we used methods of analysis and evaluation of behavioural factors determining the financial decision-making process. Consequently, we want to check how people make rational financial decisions in the modern world. The behavioural finance implies an approach in which not all participants of the market are entirely rational. The behavioural theory rejects the idea of individual rationality. Analysing and systematising this knowledge lead to optimisation the process of making financial decisions and reducing negative consequences. Moreover, models based on the classical economic theory believe that a person makes decisions oriented towards the possible optimal result. Furthermore, they assumed that a "rational person" makes this or that choice impartially -that he/she does not overestimate his capabilities. However, the reality is not so perfect: an economist (a man by nature) does not act impartially in making decisions. The Theory of Moral Sentiments is a 1759 book written by Adam Smith, a central figure of the classical political economy. This book is about human feelings, their influence on relationships between people, about attitudes towards wealth. Thus, one of the founders of economic theory as a science openly recognised the significance of human nature. Nevertheless, the classical model of economic behaviour based on the concept of "rational man" exists and develops over the years. For a long time, adherents of the classical theory responded to criticism with the approach which ignored empirical inferences. Over time, such observations have generated the new direction of researches. There appeared scientific papers describing improper decisions and their consequences in the financial sphere such as managing retirement savings, choosing a mortgage loan, investing in the stock market, corporate management of companies, "booms", "bubbles" and market crashes leading to financial crises. The behavioural approach to economics and finance emerged as a renewed approach to economic research that recognises and takes into account the human factor. In the literature, the question of corporate behavioural finance and its impact on financial decision-making is well examined and proved by various studies. Behavioural Finance: Psychology, Decision-Making, and Markets (2009) is a study written by Lucy Ackert and Richard Deaves. They focused on understanding how human behaviour influences the decisions of individual and professional investors, markets and managers. Misbehaving: The Making of Behavioural Economics (2016) by Richard Thaler, who is the Nobel Prize winner in economics in 2017 for his contribution to the field of behavioural economics. The author manages to focus on the fact that a person is not a robot who thinks solely based on laws and theories. The study presents a large number of real-life examples. Moreover, Thaler tested his theories and conclusions in real business and solved bankruptcy problems. Beyond Greed and Fear: Understanding Behavioural Finance and the Psychology of Investing (2002) is the study of Hersh Shefrin is the study of how psychology impacts finance. The author identified three areas of difference between behavioural finance and traditional knowledge of finance. While based on the conventional financial doctrine, subjects of financial relations use mathematical and statistical methods and make the right decisions, in behavioural finance, subjects can use heuristic methods of knowledge processing and, as a result, make wrong decisions.
Theoretical Aspects of Behavioural Economics and finance
Factors contributing to the emergence and development of behavioural finance The financial concepts of the 1940s did not allow for the existence of a capital market. Each Financial Contract is unique, and comparison with ordinary market rates is meaningless. In the 1950s, the stock market was not such a large capital supplier as the companies themselves. Economists at the time did not consider the stock market a suitable subject for serious research. And until the 1960s, stock prices were studied mainly by statistics (Chuvakhin, 1960) . In the 1950s, the business administration doctrine focused on the relationship between finance and accounting. Administrators of that time believed that financial and investment transactions are reflected primarily in the balance of assets and liabilities. Investors were mostly interested in the return on equity (ROE) and the return on investment (ROI). Nowadays, managers remind about maximising ROE as an all-consuming business goal. The main disadvantage of this approach is that the endless details hide the true goals of the company. Driven by new developments and mechanisms in the financial market, a scientific revolution took place in the second half of the 1950s -the first half of the 1960s. A massive increase in researches on the stock market began. Neoclassical finances started to replace 'old' finances since the latter could not answer essential questions: The law of the market is the law of a single price. Then how is it possible that different financing options can be offered to the same firm at different prices in the same market? Why are accounting, financial statements and coefficient analysis so important, although newspaper columns about stock prices are more important for shareholders? What can an unrealised profit say about a firm if investors are only interested in the yield generated by the added value and dividends relative to the market value? Therefore, a new finance paradigm should have appeared and emerged. Since the capital market is perfect, it is the only appraiser of any financial event. This statement is a paradigm of neoclassical finance since the neoclassical economy puts the perfect market at the centre of all its theories. The approach incarnated in neoclassical finances was revolutionary. There appeared new answers to old questions. Then the neoclassical theory of finance began to form. Neoclassical economics had a strong influence on financial science. It assumes the existence of a capital market perfect in any sense. Modigliani and Miller, who promoted market thinking, formed a hypothesis that is still not refuted in our time. They realised that the price reflects the attractiveness of the product for the entire population in a market economy. It means that the market value of the firm (market capitalisation) is the price of this common stock multiplied by their number in circulation in the market. Thus, maximising capitalisation is the best that managers can do for shareholders. It was a revolutionary discovery. Neither ROE nor longterm benefits are already key variables. The main thing is the added value determined by positive cash flow. Accounting profit only allows you to specify the forecast of this stream. Shleifer called the theory of an efficient capital market the "consequence of equilibrium in competitive markets with fully rational investors" (Shleifer, 2018, p. 369) . The theory of an effective market is based on three main ideas: 1) Rational investors evaluate stocks in a rational way. 2) Irrational investors may enter into random transactions that level each other and do not affect stock prices. Such investors are called noise-traders and transactions -noise. 3) Rational investors liquidate the deviations created by transactions of irrational investors of stock prices. The process of eliminating anomalies by investors is called arbitration. On this basis, it is argued that the stock market is extremely effective in reflecting information about individual companies, industries and the entire economy. News spreads quickly, equally accessible to all market participants and instantly reflected in prices. From here, you can make two conclusions: The arrival on the market of new information about the shares of a particular company causes an immediate correction of the fundamental value, and with it the price of shares; Prices will not change until new information on fundamental value arrives on the market. In the theory of rational expectations, rational expectations are identical to the forecast, which absorbed all the available information and is therefore optimal. The results of the forecast differ randomly, but not systematically, from the results of market equilibrium. Accordingly, rational expectations do not differ regularly or predictably from equilibrium results. Predicting the future, people do not make systematic mistakes. Forecast errors are random. Thus, all investors, small and large, should control risk by of income. Everybody should have a standard portfolio of assets called a market portfolio. Striving for the exact coincidence of their portfolios with the real market portfolio and realising the passive strategy of portfolio management, they will act rationally. Thus, even uninformed people can buy and sell at any time, without fear that they will be deceived by more informed market participants like insiders. An effective market protects the uninformed and therefore attracts the "masses". It is a democratic market. At the same time, market participants should be warned if the market is inefficient: "The current price may not reflect the true value, which is known only to a few informed participants." However, it is very doubtful that there would be a significant number of those who want to take part in that type of market. In an inefficient market, the public should not engage in direct transactions but should delegate to experts the right to trade at their own expense. But then access to the market is difficult, and market activity will be the privilege of a few insiders and professionals. The market in such conditions will cease to exist as a key institution of modern society. Let's try to consider a person, not a market, as the centre of the financial and economic world. People control the economy and finance. The rational and irrational components of our choice are transferred to the economy and finance and influence the process of making financial and economic decisions. The behavioural approach examines the influence of social, cognitive and emotional factors on the economic and financial decisions of individuals and institutions and the implications of this influence on market variables (prices, profits, allocation of resources). One of the most important trends in modern science is a change in the forms of rationality as a fundamental postulate of neoclassical theory. The concept of rationality and irrationality, understanding their influence on the forms of economic and financial activity, are changing radically. Scientists, based on the results of psychological research in the field of decision making, prove that people do not always behave rationally, even in their interests because of such behavioural features as self-control problems, inability to distinguish between profit and loss, difficulties in choosing between large sets of parameters, complex products, asymmetry in the perception of gains and losses, etc. In this regard, many of the results of behavioural research are relevant, and more and more research on the subject appears. In a conventional, classical economy, the assumption of rationality means that in everyday life, people compare all the alternatives that appear to each other (on any subject), and then choose the best one. Within the framework of behavioural economics, the methodological approach is initially associated with the criticism of rationality in human behaviour, whose abilities are limited due to inaccessibility of complete information and susceptibility to habits and emotions. Researchers emphasise two crucial aspects of economic behaviour: Non-rationality is a temporary state of the subject. Irrationality has systematic patterns that can be measured. Considering the rational and irrational in human behaviour, B. Hert writes: "Irrationality is a more fundamental normative concept than rationality. To call an act irrational is to declare that it should not be done; if the act is qualified as rational, then it still does not follow that it must be performed, since two (or more) rational alternatives are possible. Undoubtedly, each person, in any case, should act rationally, but this only implies that no one should ever perform irrational actions, and not at all that any rational perspective should be realised. If I have doubts as to whether this act is rational or irrational, I would rather call it rational. At the same time, it is quite possible that one would consider rational actions as such the other people would prefer to call irrational. This discrepancy, generally speaking, is unimportant, unless one decides that any concession to others is irrational. The main thing for me is not to include such a deed into the class of the irrational that someone else considers rational" (Chernyavsky, 2014, p. 22) . Causes of irrational behaviour: The juxtaposition of the present and the future. People are not predisposed to perceive reality through the prism of a long-term perspective. Abstraction of money as a concept. One of the most powerful psychological barriers of a person is the difficulty of perceiving the alternative cost of money. Behavioural economics points to regular failures in rational behaviour, systematic errors of economic participants when making decisions, and in this regard studies such phenomena as: "Herd behaviour", "Contamination by ideas", "Degraded thinking", "Collective euphoria", "Collective fear". One of the main areas of research in behavioural economics is heuristics. Heuristics is an algorithm for solving the problem. The basic idea is that human time and mental abilities are limited. As a result, a person uses a simple (heuristics) method of solving a problem to make a particular judgment or make a decision. Imagine the question of how common the name Wang is. All answer that the name is rare, except for those who live in China, where the name Wang is found very often. Considering that China is the world's populous country, on a global scale, this name is quite common. To decide how common a particular phenomenon is, people usually ask themselves how often they have met. However, this method does not work in cases where the actual frequency of repetition of an event does not correspond to the one that can be observed in its daily life (as is the case with the name Wang). Frames are another key focus of research. The frame is an abstract concept, implying "the analysis of various integrity (social, cultural) and then the assembly of structures as a set of interacting elements". Traditionally, human actions are considered as depending on the situation in which they are produced, and on the personal characteristics and qualities of the person who performs them. The concept of a frame introduces one more -the semantic angle of their consideration, speaking as a framework of social representations, within the framework of which a person determines for himself the situation in which he acts. The third direction of behavioural economic development is market inefficiency. These are decision making errors in the market that lead to various market anomalies, including incorrect pricing, inefficient allocation of resources. Three closely interrelated components are, as a rule, selected by scientists: 1) The decision of a person is usually preceded by perception, understanding, understanding of the situation and oneself in it, that is, cognitive components; 2) Subjective attitude, coloured feelings, that are emotional components; 3) The action or, conversely, its containment, that is, effectively dynamic components. The main problems of economic theory solved in the framework of behavioural economics and finance: An assumption about the axiom of independence for the theory of expected utility that does not meet reality conditions of the environment in which it is now located; The premise of the homogeneity of all goods (the effect of the initial stock: the agent appreciates the benefits that he had initially been, more than those that he can acquire as a result of the exchange; thus, non-standard situations arise around the point of the initial stock); The imperfection of human memory and computational abilities, which is fraught with failure to achieve the best possible result; The problem of discounting (in reality, agents are more focused on short-term interests than on planning the far horizon). The central question to behavioural finance research is "Why do market participants systematically make mistakes?" These errors affect prices and profits, which leads to market inefficiency. Besides, behavioural finance looks at how other participants in market relations are trying to gain from inefficiency. The main reasons for inefficiency are, firstly, the excessive and insufficient reaction to information that sets market trends (in exceptional cases, the economic bubble and the market crash) -secondly, limited attention of investors, excessive self-confidence, excessive optimism, herd instinct and noise trading. Technical analysts view behavioural economics and behavioural finance as the basis of technical analysis. Thirdly, the critical issue is the asymmetry between the decision to accumulate and save resources, known as the "bird in hand" paradox, and the fear of loss, unwillingness to part with valuable property. The trap of irrecoverable costs manifests itself in such an investor's behaviour as reluctance to sell stocks, provided that this nominally results in a loss. It can also explain why housing prices rarely and slowly fall to the level of market equilibrium in a period of low demand. The experimental financial theory uses an experimental method, in which an artificial market is created with the help of modelling software to study the decision-making process of people and their behaviour in financial markets. Nobel laureate Herbert Simon took a big part in the devaluation of the classical theory of rational choice. This scientist, relying on the work of Modigliani and Miller on the theory of the company, set himself the task "to replace the global rationality of economic man with a kind of rational behaviour that is compatible with the access to information and the computational capacities that are actually possessed by organisms, including man, in the kinds of environments in which such organisms exist () and propose definitions of 'rational choice' to become a real decision-making processes" (Simon, 1955, p. 99) . Applying the tools of the theory of rational choice with reality, Simon concluded that people make not optimal, but convenient decisions. That is, they rationally satisfy, rather than rationally optimise their needs. Behavioural economics, built on this idea, rejects the role of limited optimisation in the decision-making process. Limited rationality has become the central theme of behavioural economics and finance. In the 1990s, modern finances have undergone extensive and sophisticated empirical testing. The results are disappointing for the profession of a standard economist. The nature of the criticism of the efficient market theory can be judged by the words of Jonathan Berk: "The hypothesis of an efficient market () was an important part of the modern financial economy and remained an influential intuition, but as a formal construct, it experienced a period of its usefulness for the financial economy. I show that most forms of a hypothesis are not testable, and in tested forms, a hypothesis is easily refuted by data. The idea of unpredictable profitability is erroneous and holds back research on the financial economy There is growing evidence that some market participants receive substantial economic rent from trade. The time has come to revise the paradigm and recognise that profitability is predictable " (Berk, Stanton, & Zechner, 2008) . Economists wondered why models suddenly falter, why people do not behave in the way prescribed by the theory of rationality, which suggests that the market rate of an asset falls with its investment value. However, market prices depend on the opinion of the public, which, as we found out, is not always logical. Financial markets are too volatile because they react to a variety of news. It is how Keynes hypothesis appeared about excessive volatility. The 1980s became a period of critical academic discussions about the viability of efficient market models in the light of econometric studies of price properties, revenues and dividends. Mainly actively discussed was the issue of excessive stock price volatility concerning the forecast issued by these models. In the theory of rational economics, behavioural assumptions were almost not considered. The anomalies discovered in the course of testing the theory of rationality at worst could be considered insignificant deviations from a fundamental truth. But excessive volatility posed a greater danger to the whole concept than, for example, calendar effects on the stock market. Empirical evidence in favour of excessive volatility suggests that prices change without any fundamental reason. In the early 1980s, Schiller tested the Keynes hypothesis. He reasoned that if the stock price equals the expected present value of future dividends, as required by a rational economy, then it should not change as much as this value itself. Schiller's plan was a direct application of a simple statistical principle: a good prediction has less variance than the predicted variable. Using data on the stock prices of American companies for 100 years, Schiller compared the variance of prices with the variance of discounted dividends (after removing the trend) and found what Keynes predicted: the standard deviation of prices (forecast) was five times greater than the standard deviation of discounted dividends. This result, terrible for a rational economy, was confirmed by more sophisticated tests that took into account the non-stationarity of prices and discounted dividends (West & Shiller, 1991, p. 269) . These results inspired those who sought reasons for price volatility beyond a rational economy, particularly in behavioural finance. The collaboration of finance and other social sciences, known as behavioural finance, has greatly enhanced knowledge of financial markets. Reasoning about the achieved influence of behavioural finance, it is essential to apply the correct standards. Of course, one should not expect that these studies will open up a method for quickly and reliably extracting big money from the inefficiency of financial markets. However, the theory of an efficient market can be refuted since it can lead to a radical misinterpretation of such important events as large bubbles in the stock market. Thus, rationality is discredited, and many economists and financiers are moving away from the idea of "rational markets". Previously, many economists have ignored ideas about a person's limited ability to solve complex problems. They were quite satisfied that the existing models were not accurate enough and that the forecasts built on these models contained errors. In the statistical models used by economists and financiers, this problem is solved by the fact that the "error" of calculations is included in the equation. So, classical economists argue that errors resulting from limited rationality can be ignored. The behavioural approach suggests that such errors are not accidental, studies and systematises them, creates new methods based on the knowledge gained and applies them in practice.
The history and evolution of the behavioural economics and theory of finance Economic science gradually expands the levels of analysis of the theory and practice of the econom-ic and financial life of society. Under the influence of globalisation and informatisation of society in modern conditions, there is a complication, evolutionary expansion and the emergence of a new scientific direction of behavioural research, which means the synthesis of psychology, economics and finance, the interrelation of human psychology and the behaviour of market participants. Studies of the role of a human in the economic and financial areas of activity required a more indepth analysis of the mental and cognitive aspects of human activity, the influence of these aspects on the algorithm for making financial decisions. The model of a rational economist, conveniently placed at formal economic structures, has ceased to be satisfied due to the apparent inconsistency with reality. However, if the role of a human in shaping economic and financial relations (his psychology, preferences, mistakes, etc.) is so significant, if a person creates an economy, then the question arises: what place do objective laws occupy in the real economy, and therefore in its theoretical versions? That is, how can the behaviour of people in the economic and financial world based on predetermined exogenous circumstances be explained (predicted, foreseen)? Behavioural economics and finance in studies of recent years have shown that people's preferences are endogenous, while in the classical theory they were accepted as unchanged and externally given. It means that they are subject to change, more importantly, they can form, change "inside" the activities of people under the influence of various factors, also perceived by the behavioural model as endogenous. It became apparent that objective circumstances have given from the outside (defined by the classical paradigm as objective laws) are not capable of adequately explaining the behaviour of economic actors and their consequences, and therefore cannot adequately perform forecast functions. The relation between the objective and the subjective as the main component of the method of the economic theory requires a new approach. The distinctive characteristics of behavioural economic theory as a separate scientific direction are in the rejection of the "three whales" -the prerequisites of rationality, the pursuit of self-interest and balance. Behavioural economics is being shaped as a new direction, within which attention has been shifted from developing formal models of rational behaviour of an individual in various situations of choice to the process of their experimental and empirical testing, to determine the degree of consistency (divergence) of traditional economic theory and patterns derived from it the facts of economic activity. The behavioural approach, inherent in the activities of various subjects: from the individual and the company to the markets and regions, is systematically reproduced, complicated, and reveals the underlying motives of the agents who do not always follow the canons of the traditional economy. The first ideas of behavioural economics are reflected in the works of famous economists: Adam Smith (1723-1790), one of the founders of economic theory as a science, Alfred Marshall (1842-1924) -one of the founders of the neoclassicism, John Keynes (1883 Keynes ( -1946) ) -founder of macroeconomics as a separate science. In the works of these authors, the theory of a rational "economic man", whose purpose is to obtain benefits and income, was formed. The defining feature of the concept is rationalism and egoism; that is, the desire of the subject to maximise their benefit. The idea of "economic man" dominated economic theories for an extended period. J. Keynes first expressed the concept that irrational factors influence financial and economic processes. However, more profound studies of irrational principles were possible only at an interdisciplinary level. Thus, Keynesian theory contributed to the formation of such models that suggested too unrealistically high cognitive capabilities of the individual. Subsequently, in the controversy of new classics with new Keynesians, the latter put forward provisions on price rigidity and nominal wages as opposed to their flexibility and the possibility of a quick automatic transition to a new equilibrium. Another idea -the complete rationality of individuals (and their expectations) -was not taken into account as an object of constructive criticism. The framework of this concept did not confuse the researchers as long as the researchers were economists. Institutionalists Thorstein Veblen, John Commons, John Dewey believed that the rational approach is not the only thing that determines economic and financial behaviour, the essential elements in comparison with it are institutions, habits and customs. T. Veblen in his concept of conspicuous consumption explains the wasteful spending on goods or services with the primary goal to demonstrate consumer's wealth, and such behaviour serves as a means of achieving or supporting a certain social status. Veblen believed that individuals are constantly driven by competition, they are in a state of constant comparison their way of consuming with the other's way, determining through their consumption their position in society and the position of those around them. From the institutionalists' point of view, this behaviour is the primary determinant of consumer activity in households. Scientists have identified several effects associated with conspicuous consumption, in particular, the impact of Veblen: the increase in consumer demand because the product has a higher price that consists of two components. First, the real and prestigious prices; second, the effect of snob and effect of following the majority. At the beginning of the 20th century, a separate branch of psychological knowledge appearedeconomic psychology. The development of the industrial branch of economic psychology is associated with the name of Hugo Münsterberg, and the marketing branch that received the initial development in Western psychology was laid by Gabriel Tarde. It covers the psychological problems of exchange, distribution and consumption. One of the most prominent representatives of the marketing branch of economic psychology was the American psychologist George Katona. Behavioural game theory has made a significant contribution to the development of behavioural economics. Behavioural game theory is a direction of game theory that evaluates the behaviour of other people and suggests their further actions to make profitable decisions (Camerer, 2001) . This line of research focuses on three areas (ibid.): Mathematic theories explaining the social interaction of people at the auction and the establishment of trust between them; Limitations of strategic behaviour and cognitive ability to account for the steps of competitors; Modification of strategies in the process of training people in practice. The use of mathematical mechanics of game theory in the field of economics and finance in the second half of the last century proved to be extremely fruitful. To the greatest extent, this was manifested in those sections of the theory, the object of consideration of which is the strategic interaction of economic and financial agents among themselves in various conditions and the desire to solve the arisen conflict situation in the most optimal way. Principles of game theory: The principle of rationality; Principle of general knowledge; Principle of elimination of dominated strategies. The game theory proves that if players do not change their strategy, sooner or later they will come to some equilibrium state in which the gain can no longer increase by continuing to follow the chosen line of conduct. The game with the ultimatum (was invented in 1982). This simple game situation has attracted enormous attention of scientists precisely because the results of experimental research were significantly different from the predictions of the formal game theory, which implies that the individual follows only his interest. The game has two players and $ 100 bills for one dollar. The first participant comes to the second participant and offers absolutely any amount of these $ 100 at their discretion (a participant can offer zero). The second player has a choice: agree to the division proposed by the first player; or refuse sharing and then no one gets anything, $ 100 is taken away by the organisers of the game. Participants cannot enter into negotiations. As a rule, a rational player will settle for any amount that is more than zero -it is better to get at least one dollar instead of nothing. If he is offered a zero, he does not make a difference to agree or refuse, and he will still receive 0, regardless of his decision. Based on this, the most logical decision of the first participant will be to offer the second one $ 1 and take $ 99 to himself. However, these arguments are considered from the point of view of rationality and the theory of the games. Moreover, in real life, emotions, justice and greed will also influence the decision-making process. Zero or 1$ are offered very rarely because it is necessary to ensure that the second player agrees to the division. Most often, the first players offer the second from 30 to 50 per cent of the total. If the proposed amount is less than 30 per cent, then people begin to refuse more often (Henrich et al., 2004) . The lower the amount offered, the higher the probability of the second player refusing to share. Although, as we said above, it would be rational to agree to any amount. Some people think that the offer is too small an amount of offence and adhere to the principle "no one gets the money", thus punishing the first player for greed. Results are affected by age, culture, education, aggressiveness, lifestyle, etc. Factors such as communication and familiarity between players influence. The closer the connection, the closer the distribution of money will be to a fair 50/50 (Sanfey, 2003) . The experiments conducted by Morewedge, Krishnamurti and Ariely (2014) showed impressive results -the participants in the experiment who are intoxicated are more likely to reject unfair proposals than sober ones. Studies conducted in India in 2011 showed that the greater the amount to be divided, the fewer people refuse to distribute (Andersen, Ertaç, Gneezy, Hoffman, & List, 2011) . Nash equilibrium -such a situation in which none of the players can increase their winnings, unilaterally changing their decision. In other words, the Nash equilibrium is a position whereby the strategy of both players is the best response to the actions of their opponent. An example of the Nash equilibrium is the situation on the oligopoly market when firms have to make non-cooperative decisions. There are two oligopolistic firms in the industry -Firm A and Firm B. If both these firms could agree with each other and raise prices on their products, they would receive a high profit of $ 5 million. However, these firms are primarily competitors, and each has prerequisites to break its contract by lowering the price and thereby capturing a part of the market and getting even more profit of $ 7 million. Naturally, after such actions of an opponent, the profit of another company has decreased and will be, for example, $ 1 million. But in a real situation, trying to reduce risks and get around an opponent, each company will select low prices and make a profit of $ 3 million each, reaching Nash equilibrium (see Fig. 1 ): Pareto efficiency or Pareto optimality. A situation in a cooperative game in which a group of players gets the maximum win (equally for all who have cooperated), but any player has the opportunity to make a move unilaterally, increasing his winnings by reducing the winnings of other players. Under this rule, the right to all changes that do not cause any additional harm is recognised. In economics, a situation where Pareto efficiency is achieved is a situation when all the benefits from the exchange of the parties have been exhausted (Barr, 1992) . John von Neumann and Oskar Morgenstern, the founders of game theory, believe that the behaviour of an individual consumer depends on the behav-iour of the other participants in the relationship. It follows that in the process of interaction between subjects about the consumption and distribution of disposable income, even stable belief systems can collapse, and then other people's behaviours are chosen. The results of research by economists gave an additional argument in favour of the fact that human nature is characterised not only by a desire for material interests but also by a desire for justice and cooperation with other people. As an independent direction, behavioural economics in Western scientific literature appeared in the 1960-70s and actively developed by prominent psychological scientists Daniel Kahneman, Amos Tversky, Paul Slovic and economists George Akerlof, Robert James Shiller, Dan Ariely, Richard Thaler. In the Russian economic studies, the first researches in the field of economic psychology belong to V. Sokolinsky, A. Kitov, S. Malakhov. A fundamental contribution to the theory of psychological economics was made by B. Raizberg, who identified three main areas of research: the psychology of monetary behaviour, the psychology of labour behaviour, and managerial psychology. From the standpoint of behavioural economists, it is essential to explain the functioning of the economy and good governance, taking into account the psychological characteristics of human behaviour: changes in feelings, impressions and moods. The economic theory without taking into account subjective factors and the irrational beginning creates an erroneous understanding of economics and finance, which in practice can lead to negative results and loss.
Theoretical aspects of the decisionmaking process under risk and uncertainty In the standard model of the information economy, time and effort required to solve problems are treated as expenses. Behavioural research- ers have studied in more detail how decisions are made in situations of expanding the selection and complication of products. Some findings showed that consumers use relatively simple rules of heuristics; that is, they ignore some possible options due to a large amount of labour-intensive information. Economic and financial agents use heuristics to optimise the decision-making process in such situations, especially in conditions of time constraints, when decisions should be made quickly. In many cases, this is an effective way to achieve an optimal solution. However, these "rules" can also lead to incorrect results. Economic agents often face solutions that involve some degree of uncertainty. An obvious example is buying insurance when consumers pay a fixed amount to limit costs in the event of an accident (for example, a car accident or severe health problems). There is also a significant degree of uncertainty about the future, when economic agents borrow, make savings and investment decisions. Traditional economic models suggest that consumers, faced with the problem of choice with an uncertain outcome, evaluate the possible results depending on the probability of their occurrence and make a choice with the highest expected benefit, that is, maximise the expected utility. At the same time, consumers evaluate risky decisions in an appropriate manner. Deviations in the behaviour of economic agents explain the "Prospect theory" by Daniel Kahneman and Amos Tversky, associated with decision-making under risk. They called it a prospect theory because it highlights how people make decisions based on their prospects. The prospect theory reads as follows: There is a value function of subjective value that reflects how people value different things for themselves. There is a second component -the weighting function, which reflects the attitude of people to probability (Kahneman & Tversky, 1979, p. 263) . This model is based on three cognitive principles of consumer choice: The assessment of possible consequences is carried out in relation to the neutral point of reference, or level of adaptation. The principle of desensitisation works in assessing the dynamics of wealth. The principle of non-acceptance of losses. Consumers are ready to incur additional costs to avoid significant losses but are not ready to go to such expenses to achieve great success. Losses are experienced more than gain. Based on experimental data, scientists derived a value function, which was determined in terms of deviations from the original value. The curve is convex upward for winnings and concaves downward for losses, which means risk aversion to winnings and riskiness to lose, and the value function has a steeper slope on losses than on winnings (see Fig. 2 ). The asymmetry of the perception of winnings and losses is due to the fact that the human psyche perceives not so much the absolute value of its wealth, as its change, and the pleasure of winning is less than the disappointment of defeat. Costs always seem more significant than the equivalent income. Based on experimental research, the theory of perspective draws a paradoxical conclusion: people are more likely to take higher risks to avoid costs than to get an extra bonus with a lot of risk. For example, mass storage of money in the form of cash, despite recommendations for more rational use and support of the national economy, is explained by the natural feeling of "not taking costs", reinforced by negative experiences acquired during the times of hyperinflation, voucher privatisation and "financial pyramids". A person tends to dwell on his initial choice (anchor effect), and then make decisions that are consistent with him. At the psychological level, such a mechanism serves as self-defence against the awareness of the fallacy of the decision made. In this case, the initial choice may be random, but the subsequent line of conduct will be quite natural. An equally important effect -the effect of negative bait -is to introduce an additional choice opportunity only to capture the consumer's attention (see Fig. 3 ) in order to impose on it the most profitable purchase for the seller. In the first variant, the consumer selects according to the classical price-quality scheme, in the second, the consumer usually chooses case A from any options, since he compares his preferences to an anti-bait (-A). In other words, additional information may distract consumers from more important factors, and this can adversely affect consumer choice and make them make less profitable decisions. Moreover, such phenomena as optimism, overconfidence, availability heuristics, hindsight bias errors, turn out to be not just typical but also mass phenomena both in economic practice and in everyday life. An interesting point in the selection process is the relative probability. Depending on the context, the circles in the centre seem to be different, although, in reality, they are absolutely identical (see Fig. 4 ). Behavioural researchers emphasise that this is how consumers make choices. The effect of relativity is closely related to the demonstrative effect of consumption, consumers often imitate neighbours, friends, TV stars. The widespread use of credit cards reveals the irrationality of human behaviour. Consumers make a choice every day from several alternatives, and everyone, in principle, is able to assess the possible consequences of decisions made. People face constant trade-offs between current consumption and future consumption; Besides, the situation in which they are today depends mainly on the choices made in the past. Standard economic models of intertemporal decision-making assume that consumers choose the value of current and future consumption by discounting, which is consistent between two periods, regardless of when the consumer makes estimates. Behavioural economics argues that consumers value the present more highly than other periods. From here, they make shortsighted decisions regarding savings and loans. These consumers may, for example, take a small loan today at a higher interest rate instead of a larger loan in a year at a lower discount rate. In a general sense, the risk is an economic category expressing relationships about achieving a certain degree of success (failure) in the implementation of its goals by a business entity, taking into an account controlled and uncontrolled factors of activity. Often the risk is understood as the probability of the occurrence of any event. If such an event occurs, three economic outcomes are possible: negative (loss, damage, loss), zero, positive (win, chance, profit). One can take this probability into account by streamlining the expected impacts according to the likelihood of their occurrence. The risk we characterise as the unity of objective and subjective beginnings. On the one hand, it is generated by objective factors and exists independently of the will and consciousness of people. On the other hand, the risk is associated with the choice of certain alternatives by a specific person, which bears in itself the stamp of individuality, psychological make-up, personal motives. The occurrence of risk is determined by the probabilistic nature of many processes, unforeseen, accidental circumstances, the multivariance of economic relations into that business entities enter. People usually make decisions in the absence of complete information and certainty. In such a situation, there is a risk that the desired result will not be achieved. With uncertainty, the probability distribution of certain events is unknown. In this case, the estimated net benefit is calculated based on pessimistic, optimistic and intermediate estimates (George et al., 2017) . Depending on the individual risk attitude, people make decisions that they consider to be correct. Depending on the different attitude of consumers to risk, there are several types: Risk-takers -risk-averse, easy-to-take (assuming that the gain may be less than the initial payment); Risk-neutrals -individuals that are neutral to risk (relying on expected gains); Riskophobes -risk opponents (investing an amount strictly less than the expected income). In many cases, the risk is taken when external circumstances require it. However, most agents do not expect to get a big win and only seek to avoid losses. Often this leads to the choice of a slower, but more reliable course of action. The empirical study of strategies for repayment of multiple debts described in Psychological Factors of Multiple Debt Repayment Strategies (Gagarina & Goroshnikova, 2018) with 350 respondents reflect six different decision-making groups depending on psychological traits and attitudes to risk (see Fig. 5 ): Rational strategy is repayment of the debt, taking into account rational factors and only in that case a game is completed with a positive outcome. Semi-rational respondents try to reduce the total amount of debt, their actions are analytical but still not totally rational, and they are more risk-taking higher curiosity and flexibility. In terms of chaotic strategy, respondents have multiple errors in fulfilment of the task of paying off multiple debts; they are less open to new experience, not curious and flexible. Aversive strategy respondents reduce the total number of arrears more typical for women than for men. Respondents make some mistakes. Distributive respondents pay off all or some debts not wholly closing them. Ignorance of small numbers. Respondents with a strategy for paying off debts ignoring small amounts turned out to be more benevolent. Behavioural economics explains any economic phenomena on any scale through the lens of psychology, rationality/irrationality and behavioural mechanisms. Irrational beginning -the management of non-economic motives, irrational behaviour, persistence in delusions, spontaneous determination to act, disorder, illogic. Irrationality, as an integral feature of economic behaviour, must be taken into account when modelling. Hence, the causes of the crisis: well-established ideas, changes in attitudes, approaches to business, loss of trust, a sense of justice, ignoring the role of abuse and the sale of low-quality products -do not attach importance to stories interpreting economic mechanisms. The behaviour of agents on the market is determined by the irrational beginnings of a person, creating waves of optimism and pessimism. Behavioural economics is based on the axioms of a partial, but significant lack of understanding by individuals of the laws of market functioning, which is especially evident during periods of crisis. Individuals perceive only a small part of the total amount of information due to the complexity of the economic world. Therefore, despite the desire, they cannot make the optimal choice prescribed by theory. Testing, interpretation of economic mechanisms for any economic cycle, the ubiquity of application for different countries add a new dimension to existing models that are not able to explain the euphoria, which is replaced by pessimism. Economic reality includes many psychological variables that cannot be reflected using traditional models. Trust, honesty, optimism, irrationality, motives of behaviour, opportunism, risk, viruses, etc., are the subject of behavioural economics, which ensures the effective management of economic systems.
Irrationality in the Process of Making Financial Decisions
The main problems of decision making associated with the assumption of "rationality" The traditional approach to understanding individual decision-making is based upon classical decision theory and the rational economic model. These were initially developed in economics, and they make certain assumptions about people and how they make decisions. The rational economic model of decision-making (see Fig. 6 ) is still popular among economics scholars in suggesting how decisions should be made. However, to understand its weakness it is necessary to list its assumptions and demonstrate how they fail to match up to reality (see Table 1 ). The classical view of decision-making employs the concepts of rationality and rational decisions, in its discussions and prescriptions. Rationality is equated with scientific reasoning, empiricism and positivism; and with the use of decision criteria of evidence, logical argument and reasoning. Rational decisions are decisions which are based on rationality, that is, on a rational mode of thinking. The classical view has now been accepted as not providing an accurate account of how people typically make decisions. Moreover, its prescriptions for making better decisions have often been incorrect. Instead, contemporary cognitive research by psychologist has revealed how decisions are made based on heuristic models, judgements and tacit knowledge. Descriptive models of decision-making focus on how individuals actually make decisions. Each decision made by an individual or group is affected by several factors. Some of these include: Individual personality; Group relationships; Organisational power relationships and political behaviour; External environmental pressures; Organisation strategic considerations; Information availability (or lack of). The aim of these models is to examine which of these factors are the most important, and how they interrelate before a decision being made. One of the earliest, and still among the most influential descriptive models, is the behavioural theory of decision-making. It was developed by Richard Cyert, James March and Herbert Simon. It is called "behavioural" because it treats decision-making as another aspect of individual behaviour. For example, if a research study interviewed brokers who bought and sold shares in the stock market to determine what factors influenced their decisions, it would be an example of a descriptive approach to decisionmaking. It is also sometimes referred to as "administrative model", and it acknowledges that, in the real world, those who make decisions are restricted in their decision processes, and therefore have to settle for a less than an ideal solution. The behavioural theory holds that individuals make decisions while they are operating within the limits of bounded rationality. Bounded rationality recognises that: The definition of a situation is likely to be incomplete It is impossible to generate all alternatives Impossible to predict all the consequences of each option Final decisions are often influenced by personal and political factors. The effect of personal and situational limitations is that individuals make decisions that are "good enough" rather than "ideal". That is, they "satisfice", rather than "maximise". When maximising, decision-makers review the range of alternatives available, all the same time, and attempt to select the very best one. However, when satisficing, they evaluate one option at a time in sequence, until they alight on the first one that is acceptable. That chosen option will meet all the minimum requirements for the solution but may not be the very best (optional) choice in the situation. Once an option is found, decision-makers will look no further. The contrast between the rational decision-making described previously, and the bounded rationality discussed here is shown in Table 2 . Prescriptive models of decision-making recommend how individuals should behave to achieve the desired outcome. It makes the classical model described earlier, also a prescriptive one. Such models often also contain specific techniques, procedures and processes which their supporters claim will lead to more accurate and efficient decision-making. They are often based on obser-vations of poor decision-making processes, where key steps might have been omitted or inadequately considered. They are developed and marketed by management consultants as a way of improving organisational performance through improved decision-making. Victor Vroom and Philip Yetton developed one of the best known prescriptive models of decisionmaking, later expanded by Vroom and Arthur Jago. The focus is on decision-making situations, and on seven factors to identify the decision-making style that is likely to be most effective in any given case. It focuses on decision style, concerning how a leader decides in a given decision situation, rather than what a leader chooses to. It also concentrates on subordinate participation -the appropriate amount of involvement of the leader's subordinates in making decisions. The model consists of three main elements: Decision participation styles. Diagnostic questions with which to analyse decision situations. Decision rules to determine the appropriate decision participation style. Two key concepts underpinned the modelquality and acceptability. The quality of the decision relates to it achieving the aim; the cost of its implementation; and the time taken to implement An explanatory model of decision-making looks at what decisions were made and aims to explain how they occurred. For example, there are studies of military fiascos which examine why generals took or failed to take, certain actions. Often these made by teams have also been studied using concepts from the group level of analysis such as groupthink and group polarisation. Decisions such as whether to acquire or merge with another company have drawn upon the theories of conflict, power and politics, and have been explained at the organisational level. The judgement heuristic and biases model represents the current thinking in decision-making. The studies have highlighted the limits to rationality and introduced the concept of bounded rationality. What else might affect the individual who makes decisions? Decision-making involves choice, and choice requires both careful thought and much information. Excessive information can both overload and delay. Many managers believe that making the right decision late is the same as making the wrong decision. Hence the process is speeded up by relying on judgement shortcuts called heuristic. Decision-making using heuristics can be considered as a separate model and one that represents a further step away from the classical model. Robert Cialdini identified the decision-making biases and heuristics that could be used by individuals to influence the decisions made by others. He called them "weapons of influence": Contrast. This bias of human perception affects the way that we see the difference between things that are presented one after another. If the second item is somewhat different than it actually is. If you lift a light object first, and then a heavy object, the latter will appear than it actually is. Reciprocation. A basic norm in society is reciprocation, that is, one person must try to repay in kind in the future, what another has provided them with in the past. We are socialised from childhood to abide by the reciprocation rule or suffer social disapproval and a feeling of personal guilt. Such reciprocation leads to concession-making and allows different individuals' initial, incompatible demands to become compromised so that they finally work together towards common goals. Commitment and consistency. Commitment is a state of being in which individuals become bound to their actions, and through these, to their beliefs. Commitment sustains action in the face of difficulties. In these circumstances, it is behaviour which is being committed. It represents a visible indicator of what people are and what people intend doing. After taking an initial decision, people will adjust their attitude to make it consistent with their action, and become committed to it. Social proof. People decide what to believe or how to act in a situation by looking at what others believe and do. In case of uncertainty and ambiguity, they observe and follow others, especially those they perceive to be similar to themselves. Such similarity is defined in terms of status, social background, dress, manner or language. Market research suggests that 95 per cent of people are imitators, and only 5 per cent of people are initiators. Liking. We enjoy doing things for people we like. That liking encourages us to comply with their requests. The liking bias is so powerful that the person concerned does not even have to be present for it to The Use of Theory and Methods of Behavioural Economics in the Process of Making Financial Decisions be activated. Often, just the mention of a friend's or mutual acquaintance's name will be sufficient. Authority. Each of us has a deep-seated duty to authority and will tend to comply when requested by an authority figure. Since the opposite is anarchy, we are all trained from birth to believe that obedience to authority is right. The strength of this bias to obey legitimate authority figures comes from systematic socialisation practices designed to form in people the perception that such obedience constitutes correct conduct. Different societies vary its terms of this dimension. Scarcity. Things and opportunities that are difficult to obtain are more valued. We use information about an item's availability as a shortcut to decide quickly on its quality. As things become less available, we lose freedoms, and since we hate this, we react against it and want these things more than dimension. As individual decision-makers, we all use judgement heuristics to reduce the information demands placed upon us. Considerable mental activity is saved by summarising past experiences into the form of heuristic and using them to evaluate the present problems. Similarly, managers in organisations substitute such simplifying strategies to save having to collect complex information and analyse it. While helpful in many situations, heuristics can lead to errors and systematically biased judgements. Although the three main biases have been discussed, many other errors, fallacies and biases exist. People have ideas about the order, randomness, chance and so on. Studies have shown how peoples' judgements become biased and hence, less rational.
The main mistakes made on the stock market Adherents of the classical theories of financial markets against the researchers of irrationality in financial markets have always heard many accusations. Most of them consisted in the fact that the facts of the irrational, ineffective behaviour of operators in the market are unsystematic in nature and, ultimately, are regulated and levelled by the rest of the market. Is it really so? Does the market really have the ability to swallow a nonstandard investment activity? Or does it function with systematic errors that investors are not aware of, who are influenced by the same deviations? To understand this crucial issue, scientists in the field of behavioural finance conducted a considerable amount of research. Empirically, it was proved that when they find themselves in situations where they need to make an investment decision or to make a prediction, operators in the market tend to make the same mistakes repeated from one time to another. As such, the effects, which will be discussed below, do not have any market pegs. They take place in any stock market, be it the USA, Russia, or the other side. They are determined by the stereotypes of financial thinking, the lack of necessary diversification of financial knowledge, in some cases, the routine of the work performed, and, consequently, the narrowing of the skills used and the required amount of knowledge. All this leads to the fact that most of the operators, regardless of a national peg, get into a situation when the methodology they use is not applicable for making a successful investment decision. Many investors do not anticipate what impact they may have on the results of their investment activities. As a source of collecting, analysing and processing large amounts of information a person without loss of quality, a person does not fit very well. This position of many scientists is reflected in practice when time after time, a person shows his inability to process numerical arrays and make the right investment decisions. Some behaviourists believe that this phenomenon is caused by the fact that in the modern digital, information space, a person lives by entirely different laws than his ancestors, who lived thousands of years ago and the main choice for them lay in the area of their natural, biological needs and needs. This position may seem absurd, but it undoubtedly has the right to life, because, as practice shows, the individual is still inclined to use the mechanisms of dismemberment and simplification of information used by our ancestors. The concrete way in which these mechanisms of a person's adaptation to the problem before him are expressed and what results their use leads to will be discussed later. When carrying out financial activities, there is always uncertainty on a scale. It is almost impossible to take into account the totality of factors that determine the functioning of a process, whether it is pricing or risk identification. To be able to apply any measures to minimise the effect of uncertainty on the processes, one should calculate the probability of occurrence of a particular event. However, financial market participants use vast flows of information in their work and make their decisions according to them, and the calculation of the exact probability becomes a very laborious process. Again, if this process is accelerated by reducing the number of factors taken into account, then the final value of the probability of an event occurring will only be approximate and not capable of reflecting the real chance of a particular event occurring. In such cases, many operators take a different route to avoid substantial labour costs. Moreover, the determination of the probability for operators becomes dull, and it becomes possible to apply this method to future similar situations. The ease of application of the heuristic approach and application determines the breadth of its distribution in the financial and domestic environment. The heuristic approach is to apply any operator skills, be it his previous experience, sensations or expectations to solve problems, usually requiring the use of more complex mechanisms for its solution. The efficiency of heuristics directly depends on the situation in which it is applied. Some are quite effective; others are the opposite. Concerning financial markets, it can be assumed that heuristics are not a rational solution. There are three types of heuristics most commonly used: 1) Access heuristics 2) Representative heuristics 3) Heuristics of fastening and adaptations. Considering the heuristics of representativeness, one must, first of all, determine the essence of this concept. This heuristic is a judgment about a process or person based on stereotypes. The danger of frequent use of this heuristic is that representativeness initially ignores some factors that most often are decisive in making the right decision or judgment. These factors include: A) Misconception about the chance B) Insensitivity to basic probabilities C) Insensitivity to predictability D) Insensitivity to sample size E) The illusion of significance. Let us briefly consider these factors in turn. The misconception of a chance is because many operators believe that if there is any random process, then the generation of events by them in a long and short period will be identical. It is wrong. An example is a coin flip, an example of a classic and vividly demonstrating an incorrect interpretation of a chance. At the subconscious level, many believe that a sequence of eagle-tail-eagle-tail-tail is more likely than a tail-tail-tail-eagle-tail. Mathematically, this statement has no basis, since the subsets of a process do not always bear the characteristics of a common set. They can cause serious deviations, which are only aggravated by the small sample size. Because of this phenomenon in the financial markets, there are two effects, the effect of the "hot hand" and the "player's delusion". The first is to re-evaluate one's capabilities because of the consistent onset of operator-friendly events that were generated by a random process. As a result, this leads to de-mathematisation when making important investment decisions and losses. The second, "the player's delusion" is the opinion formed by the operator that the chance is a self-regulated value, and at certain predetermined intervals, each deviation in one direction will be levelled by movement in another. These effects are the result of not understanding the law of large numbers. Kahneman and Tversky gave this phenomenon the name of the "law of small numbers", which says that people will consider even small samples to be representative of those from which they are derived. Insensitivity to basic probabilities. The next factor that is not taken into account when using representativeness heuristics is the size of the initial probability of an event. It would seem, how can a person, evaluating various scenarios, not take into account the most fundamental information. It is empirically proven to be possible. To do this, let's consider one study (Kahneman, Slovic, & Tversky, 1999) conducted by scientists to confirm the existence of this systematic error. Were randomly recruited respondents who were asked to determine the profession of a person by his characteristics. One respondent was told that in a sample of 100 people, 70 were lawyers, 30 were engineers, and other respondents, on the contrary. For example, the following characteristic was proposed: "John is 29 years old. He is married and has no children yet. Active and purposeful, he is satisfied only with success, and constant movement forward. He is highly respected by his colleagues." As can be seen, no specific information indicating John's occupation was provided, therefore, the likelihood that he is a lawyer or engineer should remain unchanged within the previously specified ratio. As a result of the study, it turned out that both groups of respondents indicated approximately the same probability that this person is a lawyer -0.5. Initial probabilities were ignored. But it should be noted that such results were achieved only in cases when the respondent was provided with additional information that did not carry any useful information. If there was none, the respondents were close to real probabilities by their estimates. This phenomenon has been confirmed several times, both through research among ordinary respondents and among professional market participants. As a conclusion, one can say that a person objectively assesses probabilities only when he has only the basic necessary information. In a situation where the operator has any additional pseudo-information, his ability to correctly assess the possibilities of developing a certain process is reduced to a minimum. Insensitivity to predictability. Quite often, operators in financial markets are faced with the task of predicting the size of any quantities, whether it is the price of a stock, the size of profits in the future, the demand for any services. In this case, without having the ability to quickly and mathematically calculate these values, the operator gives his picture of the development of events, using the moment information about the object of forecasting. It is possible to take as an example the situation when a person has a description as issuer, without specifying specific indicators of its activity. Of course, if the description of this company is positive, any person will consider the future high profits of this company more representative than the losses. And, accordingly, vice versa. Here lies the analyst error. Based on what it is possible to give a high assessment of the future results of the enterprise, without having mathematical tools and current performance indicators? Insensitivity to sample size. One of the most common mistakes, partly described earlier, is the substitution of the law of large and small numbers in the mind of an individual. Because of this, there are often cases when errors occur in the prediction of all sorts of phenomena. Insensitivity to sample size is one of the factors that characterise the heuristic of representativeness. Take an example. If the task is to determine the likelihood that the average height of randomly selected ten men will be 180 centimetres, it is quite natural to apply in this case information about the average of these indicators among all men in general. It turns out that, statistically, the respondent denies the possible deviation of this sample, about which he has no information, and uses his empirical data to determine the probability in a situation where he is not able to reliably determine the probability. Thus, as a result of the experiment, the probability data obtained from respondents were approximately equal for samples of 10, 100, and 1000 people. In addition to this common aspect of forecasting, there is also a phenomenon called the "conservatism effect". We give an example clarifying its action. In 1968, this effect was studied by the American scientist Ward Edwards in his paper (Edwards, 1968) . And there he gave the results of the next experiment. Suppose we have two bags, in each of which there are 1000 chips, in one of them there are 700 red and 300 blue, in the other -700 blue and 300 red. If we take a coin and throw it to determine the bag that we take for the experiment, then the probability that we take the bag with the prevailing blue chips is 0.5. Then, the respondent needs to determine the probability that this bag is really with 700 blue chips, provided that of the 12 elongated chips, 8 were blue, and 4 red. Naturally, the probability that this "blue" bag is now higher than 0.5. If you give a probability of this equal to 0.7 or 0.8, then you fall into the group of the majority of respondents, and the majority is wrong in your assessment. The exact probability is 0.97. The illusion of significance. In light of the above, it is already clear that people often abuse the reduction of the outcome of the forecast to the description of the process. And the operator's confidence is the higher, the higher their similarity. Therefore, if there is an opportunity to identify the process and its result, then the operator will always do it, regardless of the quality, completeness and statute of limitations of the description of this process. The last moment is especially remarkable. Even if the operator is aware that the description of the process by which he needs to make a prediction, is lagging behind reality or contains any distortions, to save time, which he, he believes, can spend on other matters, he will often ignore this moment will give a prediction as if this information did not exist. It is this moment when the investor is so confident that he evaluates the uncertain process according to his description despite the scarcity of information on it and is called the "illusion of significance". Based on the preceding, we can conclude that it is rare when an investment decision is truly justified. Most often, the aforementioned heuristics are used for this. However, there is an effect of even deeper primitivistic of the investment choice. This effect is called the home bias. It is quite natural that in everyday life, people rely on verified information, use things they are used to. If the individual is given a choice between using two things-substitutes, one of which is well known to him, then most often the choice will be made towards the latter. This behaviour is also present in financial markets. According to statistics, market participants in different countries invest their money in the assets of the state of which they are citizens. Forming his portfolio from any national instruments, the investor naively believes that he is well-diversified, which is wrong. It would be reasonable to say that it is necessary to diversify a portfolio based on considerations of the share of different countries in the global financial market. Let us take the example of a domestic investor. According to the above principle, at first glance, it appears that it is better not to invest money in the Russian stock market. Of course, this is not the case, especially in the case of limited investment funds. However, according to the theory of portfolio investment, a set of assets from one investor cannot be called fully diversified. After all, all the papers of one national market are equally affected by country risk, which was especially clearly demonstrated by the crisis in the United States. Let this example be in some way unique, because the collapse in the United States has pulled other markets along with itself, but it is vividly demonstrating the essence of national diversification. Financial crises in different countries are not always related in time, and when constructing a portfolio of securities of the leading stock markets of the world, this will allow insuring against the loss-making of the entire portfolio. Many operators in various segments of the financial market in one way or another overestimate their ability to predict a particular event. Faced in practice with the phenomenon for which they observed and about the features of the development of which they had some idea, investors often use the ultimate significance of this process to work out a hypothesis. That is, they build their model for the development of this process, which is not always true. The investor, having obtained the result, is trying to integrate this value into the existing information system. Having laid out the processes that affected the outcome, the investor understands the weight of each of the factors in the final result. He becomes all clear, especially if he knows the methodology. The thought arises that, as it should be thought, the investor could predict the same outcome until the moment when its value would be known. This deviation is dangerous because, as a rule, such situations are used by the investor to make a "routine" out of it, that is, to use the experience gained to apply to future situations. It begins to bring negative consequences as soon as the evaluation methodology starts to lose sight of any factors affecting the outcome. This deviation at the micro-level of the investor takes place at the beginning of his activity, then it decreases to the extent of his influence on the activity, and after a more or less long period, he begins to influence his work as he gains experience in a certain area and, therefore, the investor's excessive confidence in his ability to predict the outcome of a process. To materialise the concept of "excessive self-confidence" and give it a financial justification, consider some statistical data on the US stock market and try to identify the relationship of its revaluation and financial results from this effect. In 2001, scientists Brad M. Barber and Terrance Odean (2000) conducted a statistical study of the financial activities of 38,000 households operating in the securities market to buy and sell these securities through broker companies. The sampling period was six years and was carried out from 1991 to 1997. In the standard practice for the analysis of trading activity is used an indicator called the "annual turnover of the portfolio". It is measured as a percentage and shows what proportion of the securities portfolio was sold and replaced with other securities. For example, an indicator of 50 per cent indicates that exactly half of the stock portfolio has been sold. If the figure is 250 per cent, then this means a double updating of the portfolio and the subsequent replacement of another half of the portfolio assets replaced. For a deeper structure of the study, they introduced gender and family divisions; as a result, there were four groups: single men and women, married men and women. For single men, the annual portfolio turnover is 85 per cent, for married men, 73 per cent, for mar-ried women, 53 per cent, for single women, 51 per cent. One can see how much lonely men develop a trading activity. Only 15 per cent of their securities in the portfolio per year were found by men from this group to be suitable for themselves and their investment strategy. However, one should note that since the rates are annual, it is quite possible that for the next year, those papers were replaced in the investment portfolio by others. For the interrelation of gender, self-confidence and financial performance indicators, it is necessary to consider the study of Odean and Barber (2000) concerning the connection of trading activity with the financial result. For this purpose, 78,000 households that used brokerage services were included in the sample. First of all, the meaning of trading in the stock market for a single investor is the extraction of income. At the same time, since the investor uses the paid services of a broker, then he faces the need to cover and commission. The size of the commission, of course, varies depending on the number of transactions initiated by the client. So, more deals -more commission. And the profit from resale should also significantly exceed the size of the broker's commission. And, accordingly, it is logical to assume that the yield from active trading should exceed the yield from passive portfolio management, that is, buy and hold strategies. Investors from the sample were ranked by the rate of updating the securities portfolio into five groups, 20 per cent of investors in each. The first group, with the least high rate of renewal, is about 2.4 per cent per year. The last, for the most active group, an indicator of annual portfolio renewal held at the level of 250 per cent. For these groups, the average yearly rate of return was calculated. For all five groups, the yield was close to or equal to 18.7 per cent. It turns out that all efforts to select paper, buy, and sell securities are in vain, without bringing additional returns that justify the high costs of active portfolio management. Moreover, this is a return that does not include transaction costs, taxes and other costs. After deducting payments due in all groups, it was found that the net yield for the group with the lowest renewal rate is 18.5 per cent, and for the most active, 11.4 per cent. The difference of 7.1 per cent per annum is a considerable amount, especially if you make a differentiation by the number of sums used for investment. The difference of 7 per cent per annum from $ 10,000 and $ 500,000 is incommensurable. In addition to the purely quantitative problems considered, excessive trading activity leads to difficulties of a qualitative nature. Another mini-study was conducted on the most active trading group. The average yield of the paper sold and purchased for this group was determined for four months and one year. It turned out that the paper sold would have yielded a yield of 2.6 per cent for investors from this group for four months, while the purchased one would be 0.11. In one year, the loss of profitability from such an operation is 5.8 per cent. This phenomenon suggests that the most active investors tend to sell even the most profitable of their assets in favour of low-quality securities. We would repeat that this study did not take into account the lots of purchases and sales of securities, but only the focus of the transaction and the characteristics of the papers participating in it. Potentially profitable securities in practice turned out to be assets of worse quality than those that were sold earlier. Accordingly, very often increased investment activity affects the highest-quality securities for portfolio renewal, which most negatively affects the financial result. Excessive self-confidence in the financial market is manifested in the form of a biased assessment of the risk associated with the activities of the investment operator. As a rule, there is an overestimation of the favourable outcome for an operator of an event, and an underestimation of a negative one. That is, recalling that the group of single men correlates with the rate of portfolio renewal at 85 per cent with the group of households whose returns are at the level of 11 per cent per annum, it is logical to assume that the lower returns for them turn into less risk. It is a consequence of Markowitz's theory, the classical rule of any market, higher profitability should be sought in riskier assets, and vice versa. However, is this true in this particular case? The use of the coefficient of beta, as the indicator characterising the risk of a portfolio, turns out that single men have the highest beta coefficient and single women the lowest, that is, the papers are weakly correlated with the market movement. It turns out that by buying papers with a higher risk, investors acquired low returns. It is since operators from this group had a high share in their portfolios of recently emerged companies, so-called small stocks, which, due to the characteristics of the organisation of their issuer, have a risk higher than the market average. Shares of such companies are acquired in anticipation of their rapid growth, which is not guaranteed by anyone. A rational investor seeks to limit the share of such securities in his portfolio, at least until he is convinced that a particular security paper is reliable in terms of risk and attractive in terms of its profitability. It is where the relationship between gender, risk acceptance, over trading and financial results are revealed. Apart from the need to sufficiently diversify the portfolio, single men, who share the greatest trading activity, tend to sell their most liquid and high-quality securities in favour of small company stocks, hoping for a big gain, but in fact receive higher risk for low returns, which is even lower than the strategy of passive paper holding. In itself, the knowledge that overtrading leads to a final financial result that is less than the result of passive portfolio management of many scientists plunged into shock, but in addition to this, the relationship between overtrading and gender factor further enhances the impression of the results of empirical research by scientists.
The main problems of management performance If an investor understands that his knowledge of the stock market and micro control is not enough to effectively invest in securities, more often, he transfers his money to the funds in trust management. At the same time, as a rule, they choose a fund that meets its investment preferences, the concepts of the risk/return ratio. After that, one could say that in the future, the funds will generate flows with precisely those characteristics that the founder of trust management expects. However, it is not so. Unfortunately, the work of management companies is also extremely susceptible to individual behavioural deviations, which will be proved further by the example of a similar situation in the US market. The fact is that it is in trust management that the ineffectiveness of another concept of efficient markets about investor rationality is most clearly manifested. A rational investor equally avoids risk in loss situations and profit situations. In practice, the acceptance of risk in these situations is asymmetric. For the representativeness of the experiment, Kathryn Sullivan (1997) invited to participate in an experiment to identify the characteristics of making financial decisions to 119 managers, each of whom had 20 years of experience in the financial sector and for at least six years occupied his current place. As a result, 96 financial managers took part in the experiment. Each of them had a task to choose two alternatives in five different situations, and the managers were warned that there was no right option; therefore, they ensured the originality of their investment decision during the polarisation of the experiment to the real market situation. 1) In the first experiment, the respondents were tasked to decide the condition of a probable loss of 600 thousand dollars. Managers in the condition of realisation of profits had the choice to save $ 200 in the first case or with a probability of 1/3 to save all 600 thousand, and with a probability of 2/3 to lose them. Managers in the condition of realisation of losses were asked to choose A) Lose 400 thousand dollars B) With a probability of 1/3 not to lose anything, and with a probability of 2/3 to lose everything. As you can see, the conditions for managers in terms of losses and profits are the same, with a difference only in the formulations. That is, in principle, there should be no difference in making an investment decision. However, according to the results of the experiment, 63 per cent of managers in the condition of profits chose a guaranteed profit, but in the situation of the realisation of losses assumed the risk (75 per cent). 2) The second experiment was intended to reveal the features of decision making in situations where the magnitude of profits or losses is greater. And again, the same tendency was observed as in the previous experiment, in terms of profits about 78 per cent avoid risk, in terms of losses the same 72-77 per cent assume this risk. There was again asymmetry of risk. 3) In the third experiment, the goal was to find out how the respondents react to risk, if they are given a different formulation of the problem, in terms of costs and costs. The appearance of this experiment is due to the desire to find out how much the manager links the costs with the profit from them. If he equates them to losses, then according to previous survey data, we can expect to assume the risk. If, on the contrary, conservatism regarding risk should be expected. Managers were divided into two groups: one was proposed the wording in terms of profits, the other -in costs. Profitable wording narrated about product A, which is in steady demand in the market, and which with a probability of 100 per cent will bring 420 thousand dollars in its production. There is also a product B, which with a probability of 75 per cent will bring 520 thousand dollars, and 25 per cent -nothing. The wording in terms of costs offered respondents the successful completion of the project with any choice, but in the first case it was necessary to spend an additional 420 thousand dollars to complete the project, and another with a probability of 75 per cent would require an even higher amount -570 thousand, but at the same time 25 per cent of cases no additional cost is required. Again, we see the symmetry of the construction of the problem with the difference of formulations. According to the results of the experiment, 88.2 per cent were avoided in terms of risk profits, and 62 per cent in terms of costs. Contrary to the results of the first two experiments, in unprofitable terminology, the percentage of risk managers is much lower. Costs reduce the financial result from the investment activity of the manager. But, since the experiment clearly states that the result will be positive in any case, the respondents choose the risk-free option. In this particular case, risk avoidance is excessive. Therefore, risk-taking by managers is demonstrated when the probabilities of losses and gains from the adoption of any investment policy are formulated and mathematically calculated. 4) In the fourth experiment, the task was to choose one of two investment opportunities, and both in terms of profits and in terms of costs, the net profit, in any case, was equal to 325 thousand dollars. Profitable wording was slightly modified and sounded like investment returns, and accordingly changed the wording in terms of costs. In terms of profits: there is a product A, which is in constant demand, and which will bring 575 thousand dollars in the case of the choice of its production. There is also commodity B, which has recently appeared on the market and the boundaries of the demand for it have not yet been precisely defined. However, after marketing research, it turned out that with a probability of 70 per cent in its production, you can get 665 thousand dollars, or with a probability of 30 per cent -365 thousand dollars. Both those and other options demand additional investments at a rate of 250 thousand dollars. In terms of damages, as usual, identical conditions were proposed, otherwise formulated. It was said that with any choice, the project will be successful and will bring 575, etc., the difference is only in the volume of funds required for additional investments. In the first variant, another 250 thousand dollars must be spent on the completion of the project, in the second with a probability of 70 per cent -160 thousand dollars, 30 per cent -460, etc. Again, the same conditions cause imbalances in the investment decisions of financial managers. In terms of profits, 21.6 per cent took the risk, 78.4 per cent avoided. Respectively, in terms of losses -38.2 per cent and 61.8 per cent. As you can see, most investors chose to avoid risk, that is, in the wording of profits, to get a guaranteed profit, and in the case of costs, it is guaranteed not to spend more. 5) In the fifth experiment, researchers still put respondents in the conditions for the realisation of profits and costs. In the formulation of profits, the terms were set in such a way that it is possible to choose between two alternatives, for which with a 70 per cent probability there will be a high level of market demand and 30 per cent a low one. The difference between the projects is only in absolute values of the profits that the manager will receive. In the case of high demand for the goods, he makes a profit of 465 thousand dollars, but if the demand for the goods is small, then their size will be 155 thousand dollars. Alternative commodity B has a less profitable ability depending on market conditions. With high demand, the manager receives 384 thousand dollars, with low -344 thousand dollars. The deviation of the first alternative is higher and much more to be a representative for identifying a disposition to risk. The choice of the manager of the second option is pure risk avoidance. Managers in terms of the implementation of losses were requested, at the request of state regulatory authorities, to consider two options for recycling, which are produced by the company manager. The first option implies a 70 per cent chance that it will take 465 thousand dollars; 30 per cent -155 thousand dollars. Alternative B offers a 70 per cent chance of a cost of 384 etc., 30 per cent -344 etc. As in all previous experiments, the conditions are identical, and the difference is expressed only in the formulation. According to the results of the experiment, in terms of profits, 86.5 per cent of respondents avoided risk, accepted -13.5 per cent. In terms of costs, the choice of respondents was divided approximately equally, accepted the risk of 51.4 per cent, chose to avoid, 48.6 per cent. For the first time during the five studies, the share of investors who take over and who refuse to take risks is approximately equal in terms of losses. However, this is because the losses are encoded in the form of the requirement of government agencies. In a normal situation, it is difficult to expect large profits from the recycling of your waste. It is real, to a certain extent, representative data obtained from top managers of management companies. These data, in the same way, can show us the degree of the irrationality of investment activity of funds in the USA, empiricism that cannot be interpreted ambiguously because the research conditions for managers were given the simplest ones. Asymmetry in decision making is apparent, and it can be said with absolute certainty that having, at least, approximately similar alternatives in a real situation, the financial result from the investment decision would be even worse, because, in addition to individual behavioural deviations, noise information is added, erroneously interpreted by the manager because of its uselessness. In fact, the mechanism of transferring funds to trust management, their multiplication, and the management of funds in the management company in any American, Russian (Russian in particular) and any other market is entangled in a strong network of irrationality or rather a cognitive irrationality. Let us start with the clients of mutual funds. What is the potential customer guided by when choosing the right fund? The ratio of risk/return, performance, recommendation, reputation. Of all this spectrum of factors, the first is more or less determining. Recommendations and advice are not any reliable information, because they come from a person subject to deviations and biased judgments to the same extent as the client. Information on performance indicators in various sources sometimes differs significantly, and therefore they are de facto nothing more than noise information, suitable only for compiling the most generalised picture of the foundation's activities. This behaviour has nothing to do with rational investment. Yes, and managers of funds of any market due to the nature of the activities are very susceptible to the influence of cognitive deviations. There are seven main problems of investing in various funds: 1) Forecasting. There is a tremendous amount of evidence that it is impossible to accurately predict any outcome of a process that is remote in time. And despite this, the market is fed up with all sorts of forecasts, analysts, and other unreliable information. The reason for this lies in many factors, among which are excessive self-righteousness, excessive optimism at certain stages of the financial market. Forecasting is now one of the foundations of financial activity, without which practice cannot be imagined. It turns out that despite the fact that the overwhelming majority of market participants are mistaken in their forecasts due to both the system's inability to determine the price of an asset in the most accurate way in the future, and due to the imperfection of the methodology they use, it's still everyday mass media on financial markets are filled with regular attempts to predict the outcome of any process. It may seem meaningless. In no case should we forget that the forecast is noise, no more than someone's personal opinion on what is happening, suitable only for familiarisation, the formation of his own opinion, and not for copying and applying in his investment activity. Figures 7 and 8 clearly show the quality level of analysts' forecasts for values of different nature. The first graph shows the forecasts of experts regarding inflation using the GDP deflator. The second graph shows the projections of the 10-year paper yield. It means that the respondents could not guess not only the level of the yield of the paper but also the direction of movement of this yield. In particular, 55 per cent of the forecasts for increasing profitability were differently directed with real movement, that is, in 55 per cent of cases, for a particular moment, the upward forecast coincided with a downward movement of paper. If it is difficult to predict the movement of any particular paper, even using the entire array of available information on it, it is logical to assume that the movement of an index will be easier to predict. Fig. 9 shows how this hypothesis is true. The S&P500 index on the graph is correlated with forecasts for its movement. Experts believe that the retrospective data is well tolerated for the future, as a result of which such a gap between the actual movement of the index and forecasts for two years is obtained. As one of the main factors influencing the forecasting process, one can identify excessive self-confidence. Figure 10 illustrates the results of a survey in which students and professionals associated with the stock market were asked to choose a paper in which the respondent is confident, and which, in his opinion, will grow over the next three months. As can be seen, the share of students who have guessed such papers is significantly higher than the share of stock market professionals. It follows from this that market experience is not always a guarantee of good forecast quality, and the direction of movement of securities is not as apparent as it may seem. 2) The illusion of knowledge. The financial market is always dominated by the desire to get more and more information. In scientific literature, this phenomenon is called gluttony. It is exceptionally widely believed that possessing a large volume of information allows one to be always ahead of the market and to show positive results. Of course, this is the case; an uninformed participant is in any case, less effective than an informed participant. But do not overestimate the value of owning a variety of information. It can be differentiated by its quality. Yes, and its functionality is also in question. Very often, instead of adjusting the investment decision, the additional information only increases the investor's confidence that he is right. Therefore, the principle "the more, the better" in this case is not applicable. Figures 11 and 12 reflect the results of a study on the effect of the amount of information on research participants. The purpose of the experiment was to identify how accurately a professional psychologist can determine a person's behaviour, his character, by periodical portions of information about his life, habits, and so on. At first glance, there is no connection with the financial sphere. However, returning to the results of the study, it can be noted that as the amount of information available to psychologists increases, the quality of their assumptions does not increase, that is, they still make wrong conclusions, but with each stage and with each new information their confidence is correct, it is getting stronger. Fig. 12 shows those who changed his/her opinion after receiving new information about the object of observation. The tendency to increase self-righteousness is clearly expressed. This experiment confirms the fact that additional amounts of information are interpreted not as information to the consideration or revision of their position regarding any phenomenon, but as a means of confirming it. The ability of large amounts of information of different quality to influence investment decisions is underestimated and is not sufficiently taken into account. 3) All sorts of meetings, roadshows. Many companies pay great attention to organising meetings with potential investors. However, such meetings are unlikely to be a real investment burden for the investor. There are certain issues that need to be taken into account for meetings with company representatives to bring real value. First, as mentioned earlier, more information does not mean better performance. Secondly, managers are also subject to cognitive errors, and their point of view is almost always excessively optimistic about the nature of their activities and the results that can be obtained from investing. Thirdly, for an investor who is still hesitant about any decision, the information that will be in line with his desires is dominant. Fourthly, according to research data, after such meetings, many investors admit that the representative type of the company's management played a significant role in the investor's decision making. It is a non-financial aspect that inclines many market participants towards a solution that might not be the best one on the market. And fifth, there is always the risk of receiving inaccurate information, altered, or in any other way adjusted to increase attractiveness. And not still the investor has the opportunity to check the quality of the information received. In confirmation of the above, it is advisable to bring these studies, which is carried out every quarter among the 500 largest US companies. Their CFOs are asked a lot of questions, one of which is: "Assess the potential of the economy and your company for the next quarter". In all cases, managers evaluated the potential of their company above the market, which indicates a high degree of confidence in their capabilities; that they can all replay the market (see Fig. 13 ). From this follows the conclusion that the managers of the reassessment of their management skills and financial results of the company. 4) Self revaluation. The factor is related mainly to the previous one; his/her self revaluation is more "elongated" in the time interval. Successes on any lengthy period can lead to the fact that the methodology of activity can be simplified, superoptimism and excessive trust in the manager's ability to assess and process market information, confidence in the ability to 'read' the market, outpace it will appear. To buy at the bottom and sell at the top for a long time is impossible, even having an idea about the peculiarities of the "crowd" behaviour, that is, studying behavioural finances. 5) Short term and replay. Since many operators confuse information with noise, and at the same time try to get ahead of the market, excessive trading activity occurs. In turn, it leads to a reduction in the holding period of the paper in the portfolio. On the New York Stock Exchange, for example, the average term for holding a paper in a portfolio is 8-9 months. That is, the result of such a paper is a function of the change in prices for it during this period, and this period may not be representative of the previous or subsequent ones. Such financial activities are more speculative than investment. Figures 14 and 15 show how much the holding period of securities in US mutual funds has changed over the past 90 years. If you pay attention to the 50-60s, you can see that the funds operated as long as possible at that time, and the NYSE papers were held for seven to eight years on average, as opposed to the current annual similar indicators. 6) Trustfulness. Associated with previous problems, this one is also crucial. It is necessary initially to be sceptical about the received information, changing its opinion in relation to it as other information comes from other sources confirming it. Many investors believe that they can accurately distinguish reliable information from poor-quality information. However, there are no grounds for this, and to differentiate between incorrect information and correct information, you need to have a particular array of information that will allow you to decide on the reliability of the information received. Associated with previous problems, this one is also crucial. It is necessary initially to be sceptical about the received information, changing its opinion in relation to it as other information comes from other sources confirming it. Many investors believe that they can accurately distinguish reliable information from poor-quality information. However, there are no grounds for this, and to distinguish between incorrect information and correct information, you need to have a particular array of information that will allow you to decide on the reliability of the information received. 7) Group judgment. This problem is hidden until the results of the application of group judgments in the financial sphere become clear. There is an opinion that the decision taken in the group differs in the qualitatively better side from the individual and allows achieving better results. It is a much deeper problem than it seems. Of course, with zero investor awareness, a group decision is definitely better than its unreasonable individual one. However, if the operator has some experience in working in the market, and his ability to receive information quickly and without cost is high, then the need for group actions should be carefully assessed. One would expect the group to level individual cognitive delusions, but, instead, the group only reinforces them. Gathering in a group, investors have already predetermined the task to come to any decision along the path of least resistance. The group reduces the variability of opinions. On the way to making an investment decision, the group always becomes a victim of the "anchoring effect", that is, any information that intersects in any way with their vision, they begin to interpret as confirming their predictions.
Impact of the Theory and Methods of Behavioural Economics and Finance in Practice
Use of behavioural tools in the process of making financial decisions To understand the essence of the findings of Shiller, you must first think about what determines the price of shares. Suppose a particular fund decided to buy a block of shares and keep them forever. In other words, these shares are not going to be resold, and therefore the fund can receive profit from these securities only in the form of dividends after some time. The value of the shares must be equal to the "current value" of all dividends that the fund will receive in the future after the purchase of shares, i. e. this is the amount in which the cash flow is estimated after the necessary adjustments, taking into account that the money tomorrow will cost less than the money today. Although because we do not know precisely how much profit in dividends a certain block of shares will bring, the share price is only a forecast, reflecting market expectations regarding the present value of all future dividend payments. A rational forecast has an outstanding property: as it should be on stock quotes, the forecast cannot fluctuate more than the object of the forecast. Robert Shiller, now a professor at Yale University, published the results of his research in 1981. Shiller got his results when he applied this principle to the stock market. He collected data on stock quotes and dividends since 1871. Then, for each year after 1871, he calculated what he called the "expropriational" forecast for the flow of future dividends that would go to someone who wanted to buy a portfolio of all the securities that existed at that time. To do this, he took data on the number of dividends that were paid and discounted them for the required year. Having corrected for the well-established trend, according to which, over a long period, quotes show growth, Schiller found that the present value of dividends was very stable. But stock prices, which we must interpret as attempts to predict the current cost of dividends, fluctuated very strongly. A practically flat line on the chart shows the dynamics of changes in the current value of dividends, while the bouncing line reflects the real value of the shares, both of which were corrected to eliminate the long-term effect of the increase in value over the long term (see Fig. 16 ). In his article, Shiller asked himself the question "Do stock prices fluctuate enough to explain their subsequent changes in dividends?" Based on Figure 16 , the answer to this question is positive. The results of Schiller provoked a strong reaction in financial circles and published articles. Some authors criticised the method of Schiller and his conclusions. On Monday, October 19, 1987, Robert Shiller's idea that financial markets were too volatile was confirmed. On that day, quotes dropped significantly worldwide. It all started in Hong Kong and then expanded to the West, as the exchanges opened in Europe and then in the USA. In New York, stocks collapsed by more than 20 per cent. Monday, October 19, is of critical importance because, on this day, nothing unusual happened in the field of finance or any other. No war has begun, no political leader has been killed, and nothing remarkable has happened. However, stocks fell around the world, and no one could say why. Price fluctuations continued for the next few days. In America, the S&P500 index of large companies scored 5.3 per cent already on Tuesday, jumped another 9.1 per cent on Wednesday and again fell by 8.3 per cent on Monday, the 26th. In the rational world, quotes change only as a result of the reaction to the news, and during that week the only news was that prices "jumped". If quotes are so susceptible to volatility, then they are, to some extent 'wrong'. It is difficult to argue that the price is at the close of trading on Thursday, October 15, and the price at the close of trading next Monday (which is already 25 per cent lower) can be rational indicators of true value, given the lack of news. Thus, the idea that the market price is always correct is refuted. When Shiller wrote his first article, he did not think to give explanations in terms of the behavioural approach. He merely communicated facts that are difficult to explain rationally. However, in 1984 he wrote the article "Stock Prices and Social Dynamics", in which the idea was developed that social phenomena can influence stock quotes just as they do in the fashion world. In the hypothesis of an effective market, apart from the principle that price is always correct; there is also the principle that the market cannot be replayed. Schiller's research also has a bearing on this principle and refutes it. To understand the reason, it is necessary to pay attention to some of the conclusions obtained from studies of value investment. Valuable stocks, where even securities with extremely low P/E ratios can be, show a return that exceeds the market average. You can also calculate the P/E ratio for the entire market. The question arises: does the same principle work, that is, is it possible to outplay the market if you buy stocks when they are relatively cheap and wait until they become relatively expensive? To solve this issue, Shiller preferred to use the method, which consisted in dividing the index of stock quotes (such as S&P500) by the average yield over the past ten years. He prefers this method of retrospective monitoring of profitability because it allows smoothing the temporal fluctuations that occur during the business cycle. The graph of the obtained coefficient is depicted in Fig. 17 . Evaluating the events in hindsight from the chart, you can see what the investor's strategy should be. It is important to note that when the market deviates from the historical trend, in the end, it returns to the average. In the 1970s, stock prices were fairly low but eventually recovered, and by the end of the 1990s, prices seemed rather high, but ultimately, they fell. Thus, in the long-term dynamics of the P/E index, which Shiller demonstrated, there is indeed some predictive power. However, this predictive power is not very accurate. Was Shiller's warning correct, or was he wrong? Since his warning was made four years before the market swooped down, we can say that he was mistaken for a long time before being right. This inaccuracy means that the long-term dynamics of the price/earnings ratio cannot serve as a full guarantee of profitable deals. Anyone who would follow Shiller's advice in 1996 and bet on a falling market would have suffered a loss before there was a chance for a profitable deal. The same conclusion is correct for the real estate market. The work that Robert Shiller did with Karl Case is now the widely known Case-Shiller Index of property prices. Before this index, real estate price indicators were not very reliable, since the set of houses sold in a particular month could be very different, distorting the average. At the core of the Case-Shiller Index are recurring sales of the same house, to control the type of house and its location. Long-term growth in property prices (see Fig. 18 ), where real estate price data collected by the government before 2000 is used, after which Case-Shiller data became available, so both data sources are used here. The graph shows a moderate increase in housing prices throughout the study period until the 1990s and after that a sharp rise. Besides, after a long period during which the index of the ratio of the price of buying a home to the rental price of identical housing fluctuated around the 20:1 mark, housing prices differed sharply from this long-term trend. Having this data before his eyes, Shiller warned about the danger of a real estate price bubble, which eventually happened. However, at that time no one could be sure whether it was a bubble, or something changed in the economy, as a result of which much higher purchase price/ rental price ratios were established as a new norm. It should be clarified that the inaccuracy of these passes does not mean that they are useless. When prices strongly deviate from the historical level, it does not matter in which direction the predicted value is hidden in these signals. The more the price deviates from the historical level, the more seriously these signals should be perceived. Investors should be cautious in pouring money into a market that signals of overheating, but investors should also not expect a quick profit, relying on an accurate forecast of market dynamics. It is much easier to determine the presence in the price bubble than to say when it will burst. Investors are rarely able to earn by calculating the time changes in market dynamics.
A mathematical approach to behavioural decision-making A model of investor sentiment will be considered to explain the problems of the stock market. This model is centred on beliefs. There are two stable pricing regularities: a weak price response to a single news item, for example, the earnings of stock issuers, and an overreaction to a series of specific news. In the model below, the reaction is weak, if the expected return after the good news should exceed the expected return after the bad news, and excessive, if the expected return after a series of good news is less than the expected yield after a series of bad news. Some problems in the stock market are the result of systematic errors committed by investors during the use of public information to form expectations for future cash flows. Their model takes into account two heuristics triggered by updating the original beliefs: conservatism (the tendency to underestimate the new information) and a special version of representativeness, called the "law of small numbers" (the belief that even small samples reflect the properties of the maternal population). When a company announces unexpectedly good revenues, conservatism suggests that investors react sluggishly; the stock price and subsequent returns will rise slightly. After a series of announcements of good earnings, the representativeness will cause investors to over-react, and the stock price will become too high compared to the fundamental value. The reason is that after many periods of good revenue, the law of small numbers tends investors to think (believe) that this is a company with a particularly rapid increase in revenue. Therefore, they forecast the high revenue in the future. In the end, the company cannot be average. According to the law of small numbers, if a company is average, then its revenue would be average even in short samples. Once the stock price is too high, the subsequent return is too low on average, and in the long run, reversals (price changes in the opposite direction) and the effect of scaled price ratios (scaled price ratios -rate to profit, book value to market and other price-to-cash flow ratios. Stocks with an underestimate after scaling tend to show increased returns in the future) is expected. To reflect these ideas mathematically, we developed a model with a representative and risk-neutral investor, where revenue dynamics follow a random walk process. Investors, however, do not use such a process to predict revenue. They think that at any time the revenue is generated by one of two modes: 1) Revenue returns to its average, normal value, more than in reality; good news will change badly; 2) Revenue has a more stable trend than in reality; good (bad) news will be followed by another good (bad) news. The investor believes that the revenue generated in this way varies exogenously with time and sees its task in determining which mode is generating revenue now. This approach offers one way to simulate deviations when updating beliefs. A model with trend captures the effect of representativeness, allowing investors to give the trend more weight than they should. Conservatism means that people may underestimate the latest news about good revenue in the light of past beliefs. In other words, when the news is positive, they begin to act, assuming that in the next period the news will be at least partially negative, that is, they believe in the return mode. We turn to the consequences for pricing process. Since the model participant is a representative investor, the share price is simply the investor's expected inflow (in his pocket) of discounted earnings for future periods: ( ) 1 2 2 ... , 1 1 t t t t N N P E + + = + + + δ + δ where N is revenue and 1 1+ δ is a discounting factor. In this case, the expectations are the expectations of the investor who is unaware that the dynamics of the proceeds follow the random walk process. Otherwise, because in the case of the random walk process ( ) t t j t E N N + = = , the price would be equal to / t N δ. In the model used, the price deviates from the true value of the paper, since the investor uses Source: Klyuev, 2008, p. 20 . not the random walk model, but a combination of the aforementioned modes to predict revenue. In this case, the price is satisfied by the following expression: ( ) This model shows that the interaction of the formation of investor beliefs and the true revenue model can explain two different empirical regularities -a weak reaction to certain news and an overreaction to a series of equal news. The model is based on psychological evidence and at very different values of its parameters generates the reaction of both types. 1 The model that will be given below describes the factors that influence the decision-making process of investors. In a paper written by Guney and Hussain (2007) about measuring equity mispricing, financial constraints, market timing and targeting behaviour of companies, investigated market timing theory for UK based firms. They proposed that managers increase debt (equity) issues during periods of undervaluation (overvaluation). Managers, thus, seem to time issues strategically out of necessity rather than being able to do so. Both the timing of issues and repurchasing are influenced by reaching target leverage. The evidence suggests that managers are clearly aware of the cost of being off-target and weigh this against the benefit gained from timing the market. Their initial sample comprises all U.K. firms available from 1984 to 2008. He also excludes financial firms. The final sample includes of 11,201 firm-year observations. Variables used in this model are same book leverage (BL), the net debt issues (Δdbl), the net equity issues (Δe), SIZE, Tangibility of assets (TANG), R&D and CAPEX are proxies for growth options defined as research and development expenses scaled by total assets, and capital expenditure divided by total assets, respectively. Profitability (PROF) is the earnings before interest, taxes and depreciation over total assets. The authors expand the model used by Shyam-Sunder and Myers (1999) and include a measure of valuation to proxy for timing. The model used regresses the net debt issued on the financing deficit and is defined as DEF it for firm i in year t as follows: DIV it is cash dividends, I it is net investments, ΔW it is net working capital, and C it is cash flow after interest and taxes. The sum is identical to net debt issued (Δd it ) and net equity issued (Δe it ). it it it it it it it DEF DIV I W C d e = + + ∆ -= ∆ + ∆ . The authors measure mispricing with the ratio of intrinsic value (IV) to the current market ( ) The final model is: 1 1 t equity t t e FCFE V r ∞ = = + ∑ ( ) ( ) 1 1 , 1 1 1 N t t 1 2 3 ( ) . it it it it it dbl DEF UNDVD UNDVD DEF ∆ =α + β + β + + β × + ε Thus, this model shows a strong dependence of company valuation on risks. At the same time, investors look at the above factors and make decisions about whether to invest in a company or not. Company valuation also depends on growth factors. The higher the growth of the company, the more attractive it looks to the investor, although this is not always the best option, since the growth of the company does not always reflect its stability, and the growth factors may be random (for example, inflation). In this case, the effect of an overly acute reaction can also occur investors have made an overly optimistic outlook on the future growth of some companies, but at the same time, they too low the rest. If so, then the subsequent investment of money by investors in 'good' companies and the withdrawal of money from 'bad' companies is a simple regression to the mean. Companies that have demonstrated high returns for several years in a row acquire a reputation as a 'good' company and will continue to increase. On the other hand, companies that have been lagging in the past few years are labelled 'bad companies'. Thus, a stereotype is formed at the corporate level. Indirect evidence of the overreaction of investors existed for a long time -in particular, this is a long-standing strategy of "investing in value" that Benjamin Graham was the first to practice. Graham was a professional investor and professor at Columbia University. Graham is often called the progenitor of an investment strategy in value, the essence of which is to search for securities that are priced below their real long-term value. The trick is to know how to calculate them. One of the mechanisms for determining the high cost or cheapness of the securities proposed by Graham is to calculate the price/earnings ratio (P/E), where the price per share is divided by the annual yield per share. If this ratio is high, investors pay too much per dollar of profit, and, indirectly, a high ratio is an indicator of rapidly growing stock returns to justify the current high price. If profits do not grow as fast as expected, the stock price will fall. Accordingly, in the case of a low ratio of securities, the market predicts that profits will remain low or even decline even more. If the yield starts to grow or remains the same, the stock price will rise. One study from Graham illustrates the effectiveness of his method (Graham, 1976, pp. 20-23) . Since 1937, Graham took the shares of 30 companies in the Dow Jones index for industrial companies (several major American companies) and ranked them based on the P/E ratio. After that, he formed two portfolios -one of the stocks of 10 companies with the highest P/E ratios, and the second of the stocks of 10 companies with the lowest P/E ratios. The result showed that cheap securities yielded more income than securities from the expensive price group, while with a significant margin. For the period from 1937 to 1969, $10,000 invested in cheap securities rose in price to $66,900, while the portfolio with expensive securities rose only to $25,300. If the entire portfolio of 30 companies were acquired, the yield would be $44,000. Although not explicitly, Graham essentially offered a behavioural explanation for this phenomenon. Cheap securities were unpopular or non-preferred, while expensive securities were in demand and fashionable. If you act contrary to market trends, Graham argued, you can replay the market, although not always. He drew attention to the fact that his strategy of buying the cheapest Dow Jones companies for industrial companies would not have worked in the earlier period, from 1917 to 1933, and he warned that an underestimation resulting from an oversight or prejudice could last for an excessively long time, and the same applies to overpriced prices caused by excessive enthusiasm or artificial incentives. This advice would have been worthwhile to use during the technology bubble in the late 1990s, when the cost-investing strategy worked extremely poorly since most of the expensive securities of Internet companies continued to grow in value, leaving the boring value stocks far behind. By the early 1980s, most financial economists considered the Graham approach obsolete. The simple strategy of buying cheap securities had an apparent discrepancy with the efficient market hypothesis, and Graham's methods could hardly be called modern. Data on the profitability of various companies were collected manually. Now researchers use digital databases with which they could conduct much more extensive research, and the results obtained by analysing a small number of companies in a relatively short period, as it was Graham, were no longer taken seriously. It is not that everyone rejected Graham's assertion about the value of investments in value; instead, the point was that the efficient market theory of the 1970s argued that value investment could not work. But it worked. Later, Professor Sanjoy Basu published competent research on investing in value, the results of which fully confirmed the validity of the Graham strategy (Basu, 1983) .
Barriers to the development of a behavioural approach Having considered the theoretical and practical basis of behavioural finance, we can conclude that there is still a very long way to the effectiveness of markets and rationality of investors. The system of making profits is currently built precisely on numerical imbalances, which are evident to most market participants. Few people can recognise a quality investment alternative. Behavioural finance is a new development, a lot of research is still underway, and there is a lot of work ahead in this area. However, the overwhelming majority of studies indicate the imperfection of the behavioural models of operators in the securities market and others, without putting forward specific measures on what needs to be done. Studies are indicative. In this regard, how useful can any innovative thought or theory in the financial market be, if it has no visible practical application? How to use the achievements in the field of behavioural finance, if the authors of the publications do not give specific instructions on specific measures to "beat the market" and extract increased profitability from their operations? It is worth noting that very many market participants, mainly practitioners, do not have any idea at all about the existence of a behavioural approach to the activities in which they are engaged day after day. It is these operators, or rather the features of their investment activity, that are the subject of ongoing research in the field of financial psychology. It cannot be said that the results of the research can somehow be "applied" or "used." After examining the most common market errors, the investor will have to do work to eliminate or minimise their own. The process is time-consuming, lengthy, and does not guarantee an increased financial result. It will be difficult for the operator to find out in numerical terms between the decision taken in the typical situation according to ordinary standards and when making a decision that takes into account possible behavioural deviations of the investor and with a more comprehensive methodology for analysing the effectiveness. The operator will only be confident that his decisions are based on fundamental factors that are effective in the long term. For a rational investor, it is not known how the market may behave in the future, yet the possibilities of analysis and forecasting are to a certain extent limited. Whereas irrational investors, for the most part, misuse the advancement of forecasts, operations with dubious information, and operate under the influence of many behavioural deviations that they are unaware of, or do not think about. Several factors hinder the widespread and widespread knowledge of financial psychology. Generally, they can be grouped into macro-level factors and micro-level factors. Because any direction is created to somehow influences the established order of things, this direction has to have all the required characteristics to ensure work within its framework. Behavioural finance is an element of the system that empirically denies the applicability of the theory of efficient markets with its initial assumptions about investor rationality and equal access to information for them. Research in this area, as mentioned above, are indicative. Scientists, to reveal the systematic nature of any deviation, spend a lot of time and labour on collecting statistics, analysing, processing, and grouping it, to create the depth of research, and to draw some conclusion from the total amount of work done. As a rule, there is indeed a systemic deviation in the operators' investment activity, based on statistics, the reasons for the existence of this deviation are explained in principle, and further research continues on some related issue. In the minimum number of publications you find any recommendations for the practical application of the results of the work done. This situation has developed for several reasons. First, initially, information about the presence of errors when making an investment decision does not carry any value. The specificity of the financial market is that someone wins, and someone loses. There is always an opportunity, a chance to make a profit without delving into the causes of their past failures and without drawing appropriate conclusions from them. For example, the widespread "Monte-Carlo Simulation" (Towards, 2019), when an investor neglects the law of small numbers and makes decisions based on his feelings, without even trying to assess the probability of the desired event correctly, will be of little importance if he eventually "guesses" the outcome of the event. The state of affairs could be different if scientists applied examples of their research to real-life practise to research. They explained how to counteract the influence of a particular effect, cited an approximate methodology or sequence of actions taken to avoid common deviations. However, nothing like this happens. Assessing how much time (in some cases up to 10 years) scientists spend on analysing and identifying any example of irrationality, it is doubtful that an individual investor will be able to quickly find the optimal system for analysing the event of interest, adjust the process of evaluating and making an investment decision and firmly stick to it in the future, given the various variations and modifications of the situation. Moreover, even many disparate mathematical substantiations of their research would not be enough to popularise and expand the applicability of the behavioural approach in financial markets. Works and mathematical methods should correlate with each other; you need to be able to take into account the influence of factors of one type on the investor's activities. Thus, the conclusion suggests the following: for the behavioural approach to become widespread among market participants, it is not enough to observe the mistakes of investors from different years, even if they can be systematised and generalised, and based on which you can draw some conclusions with practical value. The behavioural approach needs to have a mathematical superstructure to the mathematical apparatus of the concept of an effective market. Otherwise, unfortunately, the concept of "behavioural finance" serves as an additional source of knowledge for the investor, which should be specially noted, of an individual, and not of a legal one, for the reasons described above. The next factor that has the most negative impact on the prevalence and use of this is the degree of publicity of works studying the behaviour of investors and their delusions. The range of sources through which publications reach readers, research and various analyses of market activity is limited even in the United States to just a few publications among which The Journal of Finance, Journal of Political Economy accounts for about 40 per cent of publications, with a large number of publications directly to behavioural Finance does not apply, but they are integrated into the system of cognitive knowledge due to the possibility of correlating research results in such publications with the psychology of investing. Moreover, the practice adopted by many publications, which consists in posting long-standing articles from the journal's numbers on the website of the publication, is not applicable. Articles for some time are in the paid access. Naturally, such limited resources of information negatively affect the development of a behavioural approach in financial thought. The paper version of leading publications is difficult to get outside the USA and work in the periodical financial press, even in rare cases when the behavioural topic is touched upon, most often boils down to recommendations for more careful diversification of the portfolio and thinking about each investment decision. Due to the limited journal space, the work loses the depth of research and becomes a means of general development. Therefore, at the current moment, to be able to study any publications on the topic, it is necessary that there exists a direct interest from the investor, his time spent searching for work in the original paper or electronic version. Other sources are not well suited to be an information resource in this area, due to the limited material in most sources. There are a large number of investors and professional participants in the financial market whose interests, investment preferences and activity methodology differ significantly. It is so because these participants have different needs in the market, each in its way evaluates the quality of the paper, and everyone considers the optimal 'urgency' of the paper also differently. The active type of an individual investor, for example, as previously noted, is characterised by a high rate of portfolio renewal. This particular investor considers this type of activity to be optimal, and he will also be on the market in the future, apart from emergencies, such as crises, most likely. But with a high rate of portfolio renewal, the investor plays on short-term fluctuations in the price of the instrument, where the fundamental characteristics of the paper may not appear. Accordingly, he can get either speculative profit or loss. The relationship of this particular case with the development of behavioural finance, at first glance, is weak. However, if we recall any more or less significant work in the field of investor psychology, then it will become clear that all the studies that were conducted by scientists are long-term in nature. To identify any behavioural abnormalities, scientists usually needed time with statistical data of at least five years. How can an active investor apply the achievements of a behavioural approach to his high-intensity work in the market, while at the same time maintaining a highly updated portfolio? No, it is impossible. Of course, high intensity will lead to a higher error threshold for the investor, however, in the short-term period, the significance of the effect of cognitive deviations on the investor's activities will be minimal. As a result, an active manager will have almost the same financial result and knowledge of his own mistakes, which does not have a financial dimension. Such a disposition, as mentioned above, will occur under the assumption that the activity of portfolio management will not decrease. On the other hand, the strategy of holding a portfolio for a long time, conservatism in managing its securities in the markets of different countries is also quite common, especially in the USA rather than Russia, where the percentage of passive and active portfolio management is both qualitative and quantitative different. Quite often, investors buy a package of securities, best in their characteristics at a certain point, and then simply receive their dividends, coupon payments, and so on, without making changes in the portfolio even when the securities sharply lose in price, or a scandal is associated with the issuer In any case, a situation arises when the value of the securities, the size of the cash flow from the securities in the portfolio decreases. But this is also a case of investor psychology in the market due to its cognitive deviations. And this is not a question of choosing the most profitable tool and managing your portfolio, but a matter of selecting the option of investing temporarily free funds. As a result, not all investors find it possible, or advantageous, to use research in cognitive finance for portfolio management. Many estimate the cost of a more detailed and meticulous approach to investing higher than the profits from such behaviour in the market. Accordingly, it is not always, from the investor's point of view, to spend time studying a large number of works and publications, while it does not bring tangible, visible improvement in the financial result. It was noted above that it would take a long time for an individual investor to adapt his breeding investment activity following the experience of financial psychology research. The time lost to re-profiling its activities may be reflected in the omission of the benefits from any transaction. For a financial market participant, such as a company, or a bank, this is generally not applicable and unacceptable. Even if the participant decides to revise the principles of his activity, then it will cost a lot of time, money, you may have to update the staff of the units responsible for risk analysis and investment decisions. At this time, the participant will lose the competitive advantages associated with the reorganisation, and it is unlikely that the activity of such a participant in the 'noise' market will lead to something good, since the findings, and, accordingly, when focusing on the fundamental and noise information can be completely different. In the short run, the company will almost certainly experience problems with the selection of securities; for this, it will be necessary to form a particular selection policy. In a real market, to obtain a positive financial result, it is necessary to minimise the role of experience, retrospective data, partnership agreements and other factors on the choice of the investment object. The assessment should be carried out not only by spot (current) indicators but also take into account prospects. In general, when choosing an investment object, the mathematical-analytical approach should prevail. If the investor's cognitive deviation is a fact, then the prevention of the influence of these deviations on the investor's activities is the mathematisa-tion of processes, the use of various mathematical models that are part of different software, to which many market participants have access. Again, here it is worth noting the difference between the USA and Russian markets in this nuance. Since the share of investors in the United States is different both in its large number as compared to Russia and in their more profound differentiation among different segments of the population, it turns out that in any case, investors who do not use additional software for analysis and choosing the desired tool, it turns out that there are more such participants in the USA than in Russia. From the Russian market, this statement is supported by the fact that due to the youth of the domestic market, individual investments are made primarily by employees of companies associated with the stock market. It turns out that despite this quantitative limitation, local investors have better access in terms of percentage to the means of mathematical analysis than their foreign counterparts. Accordingly, their potential ability to choose an instrument is higher, relying on its fundamental advantages, rather than on the noise information that wraps it. This opportunity always has a positive impact on the final result of the investor, since even a loss on the instrument will be determined at the macro level, fundamentally, and the responsibility for it will rest with the investor, who in time adopted an appropriate strategy that minimises potential losses.
Conclusion During half a century history of behavioural finance it has occupied an important place in financial and economics science. Hundreds of empirical and theoretical studies that have identified systematised and described in detail all sorts of manifestations of irrationality when making decisions have proved the right to the existence of a behavioural approach to the analysis of financial activity that is impossible to ignore. Behavioural finance ideally fit into the already existing concept of financial space, revealing errors and irrationality. Most modern capital market theories are based on the so-called concept of homo economicus (rationality of economic agents), which imposes certain evident and hidden limitations on the practical side of their implementation. Studies and research in the field of behavioural economics and finance continue to appear every year, the mathematical base of the approach is getting stronger, there are more and more features, and patterns of influence of the individuality and personality of a specific market participant on the financial decision-making process are identified. The main question in this regard is how long will last the further development of the behavioural approach? Will this development ever be reflected in a more or less significant change in the current algorithm for making final decisions? If the development of behavioural finance continues, then, most likely, at some point, theorists and practitioners of the financial world will need to integrate cognitive finance into their activities in some way, to supplement primary educational literature with behavioural finance. The generally accepted rational model of stock markets describes not how investors make decisions in reality, but how they should do it. That is the hypothesis of an effectively functioning market abstracted from the personal characteristics of the participants in this market and the psychology of crowd behaviour. In contrast to this point of view, a different perspective on the behaviour of investors prevails in cognitive (behavioural) psychology. It is believed that the process of human decision-making is directly influenced by subconscious factors. Among them are such as mental models (heuristics), emotions, crowd influence, etc. The theory of behavioural finance has proven and postulated that in the process of forming their future expectations (that is, the market vision), market participants often resort to not exact mathematical calculations and independent analysis of available information but use the so-called rule of thumb or heuristics, that is, simplified solution strategies complex problems with limited information. Such behaviour in the financial and economic markets in some situations may be justified and even necessary. However, in most cases it leads to preconceived future expectations and irrationality of actions. Not all economists have abandoned their commitment to efficient market theory. But the behavioural approach is now taken seriously. On many issues, the dispute between rational and behavioural has been central to publications on financial economics for more than two decades. Focusing on the data is what makes this debate continue to be valid and productive. Most economic theories do not proceed from empirical observations; instead, they are based on axioms of rational choice, regardless of whether or not these axioms have any relation to what we observe in our daily life. The theory of rational behaviour cannot proceed from empirical data because entirely rational people do not exist. The combination of facts that are difficult or impossible to reduce to the theory of an effective market, behavioural research -all this contributed to the fact that the field of finance has become an area in which the statements about the invisible hand have undergone a severe review. Financial markets as an area of research are an indication of how a data-based economy can lead to the development of a new theory. The discovery begins with the detection of anomalies. It is impossible to call the completed work on the formation of a new version of finance, based on empirical data, but it is rapidly developing. However, the principles of behavioural economics and finance can be applied with advantage now. In a rational world, investor makes financial decision to maximise their risk-return trade-off. They have all the information they need on estimated return and risk, and they make their choices according to this information. In traditional theories of financial investment decisions are based on the assumption that investors act rationally. It means that their behaviour is rational -so they earn returns on the money they put in stock markets. It is essential for investors to have rational behavioural patterns to become successful in the stock market. Rational behaviour is also required to be financially successful and to overcome tendencies. The modern theory of investors' decisionmaking suggests that investors do not always act rationally while making an investment decision. They deal with several cognitive and psychological errors. These errors are called behavioural biases and are there in many ways. Thus, summing up, it is worth to say that today, it is simply impossible to deny the existence of a clear relationship between the price movements of the markets and the psychology of their participants. Only by taking into account and comprehensively studying the peculiarities of the behaviour of people as the leading market participants, can we build any kind of holistic and really functioning models of financial decision-making. People will remain people after thousands of years, so the study of human nature and human behaviour now is of paramount importance and is, in fact, the formation of a kind of foundation for the further construction of the building of behavioural science. Fig. 1 . 1 Fig. 1. Oligopoly pricing. Source: The author.
Fig. 2 . 2 Fig. 2. The utility function in behavioural economics.Source:Tversky & Kahneman, 1992, p. 313.
Fig. 3 . 3 Fig. 3. The anti-bait effect when selecting from several alternatives.
Fig. 4 . 4 Fig. 4. Illustration of the principle of relativity in the selection process. Source: https://epee.hse.ru. Accessed 03 April 2019.
Fig. 5 . 5 Fig. 5. Representativeness of strategies.Source:Gagarina & Goroshnikova, 2018, p. 60.
Fig. 6 . 6 Fig. 6. Rational economic model of decision-making.Source:Buchanan & Huczynski, 2017, p. 312.
Fig. 7 . 7 Fig. 7. US GDP deflator and forecasts. Source: Bloomberg.com. (2019). About US Real GDP. Available at: https://www.bloomberg.com/quote/EHGDUSY: IND. Accessed 5 March 2019.
Fig. 8 . 8 Fig. 8. Consensus one year ahead bond yield forecasts and reality (per cent).
Fig. 9 . 9 Fig. 9. S&P500 Index Forecast.Source:Russo & Schoemaker, 1989, p. 97.
Fig. 10 . 10 Fig. 10. Average Accuracy and confidence considering stock selection (%).Source: https://www.drkwresearch.com. Accessed 8 Apr. 2019.
Fig. 11 . 11 Fig. 11. Increase confidence as the volume of available information increases. Source: Slovic, 1991.
Fig. 12 . 12 Fig. 12. The share of experts who change their opinion each round.Source: Slovic, 1991.
Fig. 13 . 13 Fig. 13. Optimism over the economy and own firm (%). Source: Cfosurvey.org. (2019). Duke CFO Global Business Outlook. Available at: http://www.cfosurvey.org. Accessed 8 April 2019.
Fig. 14 . 14 Fig. 14. Average holding period of US mutual fund investors (years).Source: Bogle, 2005, p. 37.
Fig 15 . 15 Fig 15. The average holding period of NYSE listed stocks (years).Source: Graham, Harvey, & Rajgopal, 2004.
Fig. 16 . 16 Fig. 16. Are Stock Markets Too Volatile?Source: Shiller, 1980, p. 292.
Fig. 17 . 17 Fig. 17. Long-Term Stock Market Price/Earnings Ratios. Source: Home Page of Robert J. Shiller. Available at: http://www.econ.yale.edu/~shiller. Accessed 9 May 2019.
Fig. 18 . 18 Fig. 18. Long-term dynamics of housing prices and rental prices in the US.
Table 1 1 Rational economic model assumptions and realityThe Use of Theory and Methods of Behavioural Economics in the Process of Making Financial Decisions it. The acceptability of the decision refers to subordinates and anyone else either affected by the decision or who has to implement it. Leaders and managers generally select the highest quality decision that is acceptable. Assumption Source: Buchanan & Huczynski, 2017, p. 314.
Table 2 2 Rational decision-making and bounded rationality contrastedThe author, based on Kahneman, Diener, & Schwarz, 2003. Rational decision-makers Bounded rationality decision-makers Recognise and define a problem or opportunity Reduce the problem to something that is easily thoroughly understood Search for an extensive set of alternative courses of Develop a few, uncomplicated and recognisable action, gathering data on each solutions, comparable to those currently being used Evaluate all the alternatives at the same time Evaluate each alternative as it is thought of Select and implement the alternative with the most value (maximise) Choose the first, acceptable alternative (satisfice) Source:
FCFE t is free cash flow to equity at time t, and r e is the cost of equity. FCFE is the sum of net income plus depreciation minus change in non-cash working capital minus capital expenditure minus principal repayments of debt capital plus new debt issued. Where g is the long-term FCFE growth. Given that FCFE occurs throughout the year, we make adjustments as follows: equity = V 1 N t = ∑ ( ) 1 t t e FCFE r + ( ) 1 e r + = 0.5 1 FCFE + ( ) ( ) 1 1 e t e g r r + + 0.5 . Misvaluation = it it MP IV equity = = t t t e FCFE ∞ V r = = + ∑ ∑ t FCFE TerminalValue N e e r r + + + price (MP). Intrinsic value is measured as follows: TerminalValue = ( ) ) 1 g r g N e FCFE ( + - .
The Use of Theory and Methods of Behavioural Economics in the Process of Making Financial Decisions
Source: The author.The Use of Theory and Methods of Behavioural Economics in the Process of Making Financial Decisions
|
10.3201/eid2902.221674
|
cc-by
| null | null |
openalex
|
M onkeypox virus (MPXV) is a zoonotic orthopox virus. An outbreak of MPXV infections emerged in the spring of 2022 outside Africa, mainly in Europe and the United States, such that on July 23, 2022, the World Health Organization declared the outbreak to be a public health emergency of international concern. During this outbreak, MPXV spread has disproportionately affected gay or bisexual men or other men who have sex with men, which suggests transmission through sexual or intimate contact. However, in August 2022, we observed intrafamilial transmission of the virus to all members of a family (father, mother, and 2 children) in France. The father, a 30-year-old-man, showed a few papular pustules on his body, including his penis. The pustules began appearing on July 17, 2022. A pustule sample was tested for MPXV and showed a positive PCR result. The wife of the man showed a few pustules on August 2 that were later confirmed to be positive for MPXV by PCR. She had no mucous signs. Both persons were HIV negative. The couple and their 2 young daughters went on vacation to a campsite in southern France on August 6. Their 4-year-old daughter had a fever (temperature 38C) and a skin eruption that began on August 5, which consisted of 3 types of lesions: an umbilical pustule (Figure , We report intrafamilial transmission of monkeypox virus to all members of a family (father, mother, and 2 children). Case reports in young children have been extremely rare during the 2022 mpox outbreak. Their clinical signs were mild, and clinical diagnosis would be difficult without knowledge of the father's monkeypox virus infection. On August 9, her 7-year-old sister showed ≈10 asymptomatic micropapular pustules on a discrete erythematous basis (Appendix Figure 1 , https://wwwnc. cdc.gov/EID/article/29/2/22-1674-App1.pdf). She had no fever, mucosal lesion, or lymphadenopathy. The family was residing at a campsite in southern France, where there is a high burden of Aedes albopictus mosquitoes. They were suspected of having either MPXV infection or Aedes sp. mosquito bites. A skin scraping from the skin of a micropapular pustule was positive for MPXV by PCR. For all family members, only 1 lesion/person was sampled. Onset symptoms and positive PCR results are shown in the timeline (Appendix Figure 2 ). The family received isolation instructions and returned home on August 11. The outcome was favorable for all 4 persons. There were no secondary cases at the campsite. During the ongoing outbreak, cases of MPXV infection in young children have been extremely rare. US data from the Centers for Disease Control and Prevention as of October 7, 2022, reported 26,577 cases in 28 patients (0.1%) <12 years of age (1, 2) . Those data were confirmed in Europe. As of August 3, 2022, among 4,663 laboratory-confirmed cases of MPXV infection reported in Spain, only 4 (0.1%) were in children <4 years of age (7, 10, and 13 months, and 3 years) and 12 were in adolescents 13-17 years of age (3) . As of August 23, 2022, a total of 41 countries in the World Health Organization European Region have reported 21,098 cases, of which 15 (0.07%) were in persons <15 years of age (4) . Intrafamilial transmission to all family members including both children, also appears extremely rare in the 2022 outbreak. We assume that the transmission route was through household direct skin-to-skin contact with their parents. The clinical signs shown by the children were mild, and clinical diagnosis would be extremely difficult in the absence of knowledge of the MPXV infection of the father. Case reports in young children have been extremely rare in this outbreak (5-7). Thus, a dermatologic description is lacking in this population. The 2 children in this report had mild general signs. Skin lesions consisted of a few umbilical pustules or papulopustules similar to those reported (8) (9) (10) . However, the children also had discrete micropapular pustules on an erythematous basis similar to mosquito bites and a faint erythematous rash. Such skin lesions have rarely been reported in children or adults (8) (9) (10) .
Dirofilaria immitis in Dog Imported from Venezuela to Chile Cristian A. Alvarez Rojas, 1 Beatriz Cancino-Faure, 1 Pablo Lillo, María Luisa Fernández, Alejandro Piñeiro González, Alonso Flores Ramírez D irofilaria immitis, a species of zoonotic parasitic nematode transmitted by mosquitoes, causes canine dirofilariosis. These nematodes are usually found in countries with temperate and tropical climates and are endemic throughout Europe and in the southeastern regions of Asia and Africa (1). In the Americas, D. immitis nematodes are present in all countries and territories except Chile and Uruguay (2). We report a case of a female dog born in Venezuela and imported to Santiago, Chile, where she lived for 2 years before having D. immitis infection diagnosed in January 2022. The dog was a 5.5-year-old Shar-pei who was brought to 2 veterinary clinics in Santiago during December 2021-January 2022. The initial cause of the consultation was vulvar discharge evolving to vomiting, melena, and general discomfort. Initially, the dog's health improved after treatment with enrofloxacin (5 mg/kg). However, the animal's condition deteriorated after 1 week (in January 2022). Ultrasound examination showed the presence of a fetus for which no heartbeat was detected. Blood work showed severe anemia, kidney failure, and the presence of microfilariae at the blood smear examination. An echocardiogram examination showed signs compatible panel A), papular pustules on an erythematous basis (Figure, panel B), and a disseminated faint erythematous macula (Figure, panels C, D). She also had a bilateral conjunctivitis but no lymphadenopathy or mucosal lesions.
Figure . . Figure. Monkeypox virus lesions for the 4-year old daughter in a family (father, mother, 2 children) infected with the virus, August 6, 2022. A) Umbilical pustule on pulp of the finger; B) papulopustule on the ankle; C, D) faint erythematous rash on the thighs.
Author affiliations: Escuela de Medicina Veterinaria, Facultad de Agronomía e Ingeniería Forestal, Facultad de Ciencias Biológicas y Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile (C.A. Alvarez Rojas, P. Lillo); Laboratorio de Microbiología y Parasitología, Departamento de Ciencias Preclínicas, Facultad de Medicina, Universidad Católica del Maule, Talca, Chile (B. Cancino-Faure, A. Piñeiro González); Hospital Veterinario Mi Mascota, Santiago (M.L. Fernández); Sociedad Chilena de Cardiología Veterinaria, Santiago (A. Flores Ramírez) DOI: https://doi.org/10.3201/eid2902.221427
Emerging Infectious Diseases www.cdc.gov/eid Vol. 29, No. 2, February 2023
Emerging Infectious Diseases www.cdc.gov/eid Vol. 29, No. 2, February 2023
These first authors contributed equally to this article.We report a case of Dirofilaria immitis nematode infection in a dog imported from Venezuela that had been living for
years in Santiago, Chile, where this parasite had not been reported before. Our findings warrant surveillance for all dogs imported to Chile, given that suitable conditions exist for establishing this parasite.
|
10.22329/tclr.v1i2.7931
|
cc-by
| null | null |
openalex
|
One of the most pervasive and longstanding problems in the practice of mutual legal assistance in criminal matters between states has been 'form of evidence'specifically, can the requested state provide evidence in such form as will be useful and admissible under the criminal evidence laws of the requesting state? It tends to be common law states that have difficulties with admissibility of MLAT-sourced evidence, and these often develop 'work-arounds' in their laws which attempt to relax admissibility standards. Canada is one such state, but a series of recent prosecutions has revealed judicial resistance to the tools employed. This note examines these cases and suggests some lessons they contain for broader practice.INTRODUCTION Once touted as 'the fastest growing business in the criminal justice field,' 1 mutual legal assistance (MLA) in criminal matters is now a well-established mode of inter-state cooperation in the suppression of crime. 2 It is a feature of bilateral, 3 regional, 4 and global regimes that are designed to facilitate evidence-gathering and prosecutions for offences that have transnational aspects. 5 It certainly has its modern-day travails, in particular the concern that MLA arrangements are too slow-moving and administratively unwieldy to accommodate investigational needs in cyber-crime cases and cases involving electronic evidence generally. 6 Despite this, however, it is a fairly ubiquitous tool in the criminal cooperation toolbox and likely to remain so for the foreseeable future. MLA is, of course, designed to allow the authorities of a state on the territory of which evidence can be found (the 'requested state') to gather and send evidence to a foreign state which requires it for use in a prosecution (the 'requesting state'). This addresses the sovereignty concerns which would arise from police or other officials in one state attempting to unilaterally gather evidence on the territory of another, which would offend the prohibition on extraterritorial enforcement jurisdiction. From early days, however, MLA practice has experienced irritants, a major one being form of evidenceto wit, the requested state might be perfectly willing to gather and send evidence to the requesting state, but potentially not in a form that is ideal or even acceptable to the latter, from the point of view of admissibility in court. Managing these problems can require busy and often-overworked officials to make tricky inquiries into whether gathering or compiling the evidence in a way that suits a foreign state's law is possible under their own law. 7 The overall issue is that the criminal justice systems of states do not 'speak each other's language' they are not interlocking or compatible enough. 8 One flashpoint for this kind of conflict is hearsay evidence, defined for current purposes as evidence of statements made by individuals out of court, introduced at trial to prove the truth of the contents of the statements. 9 Common law states have historically taken a fairly restrictive approach to the admissibility of hearsay, typically by making it presumptively inadmissible subject to exceptions. Civil law states and states with otherwise inquisitorial trial systems, by contrast, tend to take a very relaxed approach to admitting hearsay and tend simply to give hearsay statements the weight to which the court feels they are entitled. 10 Accordingly, police investigational methods in civil law states (and those with other systems, as well) might very well generate a fair bit of hearsay evidence, and it can be difficult for those states to produce evidence under an MLAT request that is as useful as the requesting state would like. Criminal law is intrinsic to state sovereignty and, as has often been said, states tend to be quite chauvinistic about their own versions and suspicious of others. 11 Needless to say, states do not always take kindly to requests to tailor their criminal procedure to the formal requirements of foreign legal systems, and sometimes simply cannot accede to such requests under their domestic constitutional arrangements or lower-level laws. The resulting practical burden has been on common law states to formulate work-arounds for hearsay evidence received under MLAT. 12 As discussed in further detail below, Canada is one of those states; its domestic statute implementing its various MLATs contains provisions meant to ease the admission of hearsay that is received via a treaty request. Of late, however, Canadian Crown prosecutors have encountered difficulties with this machinery, in the form of a series of court decisions holding that the statutory provisions have gone too far in relaxing admissibility standardsto the point that they render trials unfair and thus fall afoul of the constitutionally-protected rights of the accused. This short article will offer a review of these recent developments in Canada, as a means of illuminating relevant state practice in the often-murky world of inter-state criminal cooperation generally and MLA practice in particular. The legal findings discussed are, to some extent, driven by the peculiarities both of Canada's hearsay law and the wording of the statute implementing the MLA obligations. Nonetheless, there are instructive points of general application that emerge.
CANADIAN HEARSAY LAW While this article cannot offer detailed observations on comparative criminal evidence law, it is worth highlighting the point that among the two dominant legal systemscommon law and civil lawcontrasting approaches are taken to trial evidence. Civilian systems tend to be quite generous towards the admissibility of evidence, with the fact-finder (typically a judge or investigating magistrate) simply assigning items of evidence the significance they are felt to deserve. In common law states, by contrast, evidence law generally contains a fairly rigid set of rules that exclude or carefully manage certain kinds of evidence due to concerns about its probative value; classic examples are character, opinion and, most relevant here, hearsay. Importantly, the common law imposes a division of labour between the 'trier of law' (the judge) who makes decisions as to whether evidence is admissible, and the 'trier of fact' (the jury, or the judge in a judge-alone trial), which makes the findings of fact based on its evaluation of the admissible evidence (usually referred to as 'weighing'). The restrictive admissibility rules are primarily designed, then, to "protect" the trier of fact from the prejudicial effect that might result from admitting suspect evidence. For the purpose of admitting documentary evidence, the common law traditionally maintained a distinction between authentication (the question of whether evidence proffered by a party truly is what that party claims it to be) and admissibility (will the evidence be 'admitted,' that is, placed before the trier of fact to be used in its determinations regarding the facts of the case). Canadian evidence law essentially has all of the features described above. In order to explain comprehensibly the MLAT case law being discussed here, a basic review of Canadian hearsay law is required. 13 As noted, Canada is a common law state and Canadian evidence law is almost entirely sourced in the common law, which is lightly modified by statute. Consistently with its common law heritage, Canada takes a restrictive approach to hearsay and has historically treated hearsay as presumptively inadmissible, subject to certain welltrodden exceptions. Beginning in roughly 1990, however, 14 the Supreme Court of Canada reshaped hearsay law along two axes: the 'principled approach' to evidence law, and the constitutionalization of the right to a fair trial. Each of these bears brief explanation. The 'principled approach' rested on the Court's recognition that the traditional approach to admitting evidenceessentially, rules barring admission which were subject to exceptionswas overly crude because it would not always respond to the factual situations at play in individual cases, and could result in both under-and over-exclusion of evidence. 15 The better course was to formulate higher-level 'principles' that would illustrate both the potential probative value and prejudicial effect of particular types of evidence; the detailed balancing of these was properly left to the trial judges charged with admissibility decisions, for evaluation in a manner suited to the case before them. So far as hearsay went, the Court drew on the writing of Professor JH Wigmore and distilled the principles of admitting hearsay into twin criteria of 'necessity' and 'reliability.' Though presumptively inadmissible, a hearsay statement can be admitted if it is necessary (the original maker of the statement is unavailable or evidence of similar value cannot be introduced) and reliable (the statement was made in circumstances that would tend to make it safe for the trier of fact to rely on it as being true or accurate, or, there are sufficient procedural means available to test the reliability of the statement at trial). Through this lens, the traditional exceptions (for example, dying declarations, spontaneous utterances, and so on) could be seen as specific applications of necessity and reliability; moreover, the Court fashioned a residual exception under which a hearsay statement not fitting a traditional exception could nonetheless be admitted if it was necessary and displayed indicia of reliability. As the Court developed its approach, it was explicit that this more nuanced and rigorous approach to hearsay admissibility was driven in part by fair trial considerations. The right of accused persons to a fair trial, which had always been enjoyed at common law, became a constitutional norm with the 1982 introduction of the Canadian Charter of Rights and Freedoms. 16 The Court's overall approach has been to use this Charter requirement (specifically found in sections 7 and 11(d) of the Charter) to discipline the law of evidence, on the intuitive notion that defective or unfair admissibility rules increased the danger of unfair trials. Regarding hearsay, as early as 2000 the Court noted that: 'It would compromise trial fairness, and raise the spectre of wrongful convictions, if the Crown is allowed to introduce unreliable hearsay against the accused, regardless of whether it happens to fall within an existing exception.' 17 In its 2006 decision in R v Khelawon, 18 which is still the leading decision regarding the admissibility of hearsay, the Court provided the specific linkage: The trial judge's function is to guard against the admission of hearsay evidence which is unnecessary in the context of the issue to be decided, or the reliability of which is neither readily apparent from the trustworthiness of its contents, nor capable of being meaningfully tested by the ultimate trier of fact. In the context of a criminal case, the accused's inability to test the evidence may impact on the fairness of the trial, thereby giving the rule a constitutional dimension. Concerns over trial fairness not only permeate the decision on admissibility, but also inform the residual discretion of the trial judge to exclude the evidence even if necessity and reliability can be shown. As in all cases, the trial judge has the discretion to exclude admissible evidence where its prejudicial effect is out of proportion to its probative value. [.] Prior to admitting hearsay statements under the principled exception to the hearsay rule, the trial judge must determine on a voir dire that necessity and reliability have been established. The onus is on the person who seeks to adduce the evidence to establish these criteria on a balance of probabilities. In a criminal context, the inquiry may take on a constitutional dimension, because difficulties in testing the evidence, or conversely the inability to present reliable evidence, may impact on an accused's ability to make full answer and defence, a right protected by s. 7 of the Canadian Charter of Rights and Freedoms. The right to make full answer and defence in turn is linked to another principle of fundamental justice, the right to a fair trial. The concern over trial fairness is one of the paramount reasons for rationalizing the traditional hearsay exceptions in accordance with the principled approach. 19 The fundamental take-away point, then, is that the inappropriate admission of hearsay endangers the accused's right to a fair trial, a fundamental right which is protected under the highest law of the land; and that the admissibility of hearsay is primarily based on screening it for its necessity and reliability. From a remedial point of view, in Canadian constitutional law any statute that breaches a Charter right, on its face or in its operation, is subject to being 'struck down,' that is, ruled to be unconstitutional and declared to be 'of no force and effect.' 20
CANADIAN MLAT LAW Canada is party to approximately 58 bilateral MLATs, and also to a substantial number of multilateral criminal suppression conventions which contain MLA provisions, such as the UNTOC. 21 Canada takes a transformationist approach to the implementation of treaty obligations, and all of its MLA obligations are implemented via the Mutual Legal Assistance in Criminal Matters Act, 22 a federal statute with application across all criminal and federal regulatory proceedings. For cases where Canada is the requesting state, part II of the Act contains a small suite of provisions designed to ease the admissibility at trial of foreign-gathered records or 'things,' as well as presumptions of admissibility for documentation which purports to authenticate the records or things. Of particular interest here is section 36, which provides: in evidence by reason only that a statement contained in the record, copy, affidavit, certificate or other statement is hearsay or a statement of opinion. Probative value (2) For the purpose of determining the probative value of a record or a copy of a record admitted in evidence under this Act, the trier of fact may examine the record or copy, receive evidence orally or by affidavit, or by a certificate or other statement pertaining to the record in which a person attests that the certificate or statement is made in conformity with the laws that apply to a state or entity, whether or not the certificate or statement is in the form of an affidavit attested to before an official of the state or entity, including evidence as to the circumstances in which the data contained in the record or copy was written, stored or reproduced, and draw any reasonable inference from the form or content of the record or copy. Foreign For present purposes, on its face section 36 attempts to accomplish two things: under subsection (1), the contents of documents ('records') obtained from foreign states via MLAT are not inadmissible simply on the basis that those contents amount to hearsay; and subsection (2) provides that the court may take a wide variety of evidence into account as it determines what use it will make of the document, that is, what 'probative value' it will assign to it as it weighs the evidence. It is not illogical to read this as a direction that foreign-sourced hearsay is automatically admissible and that all of the considerations that might normally go to admissibility are instead assigned to how the trier of fact weighs the evidence. Indeed, surveying the case law reveals that it is exactly this interpretation that the Crown has urged on the courts for many years. However, this position has mostly been staunchly resisted by the courts, 23 essentially on the basis that the admissibility of evidence is based in the inherent jurisdiction of the courts and not lightly removed by Parliament; and in particular that the courts always maintain the discretion to exclude evidence the probative value of which is outweighed by its prejudicial effect, since to provide otherwise would endanger trial fairness and thus run afoul of sections 7 and 11(d) of the Charter. 24 Nonetheless, the Crown has continued to make the argument for the automatic admissibility of MLAT-sourced hearsay, based on the wording of section 36. This strategy backfired spectacularly in a complex human smuggling case in the Canadian province of British Columbia, to which I will now turn.
WHEN CHICKENS CAME HOME TO ROOST: CHRISTURAJAH/RAJARATNAM In R v Christurajah, 25 the four accused were tried for allegedly smuggling undocumented migrants from Thailand to Canada. 26 The Crown sought to adduce in evidence a number of records received from the government of Thailand via an MLAT request, including police records regarding the arrests of three of the accused in Bangkok, as well as travel, purchase and ownership documents relevant to the investigation, all attached to affidavits executed by a Thai police investigator. The documents contained both a great deal of hearsay and some statements of opinion. The accused made a motion to have the court declare that sections 36(1) and (2) of the Act were unconstitutional and of no force and effect, on the basis that they did not allow for appropriate screening of hearsay evidence and thus violated the right to trial fairness under sections 7 and 11(d) of the Charter. In response, the Crown argued that the impugned sections simply removed the hearsay or opinion rules as the basis for excluding the evidence, and that it could still be excluded if it would breach another rule of evidence (for example, character), or if its probative value outweighed its prejudicial effect for some other reason than that it was hearsay or opinion. Moreover, section 36(2) gave the trier of fact some discretion over how to weigh the evidence, and from a policy point of view it was important that the Crown have 'effective tools for combatting international crime.' 27 The trial judge, Ehrcke J, accepted the defence position and granted the motion. Reviewing the hearsay law outlined above, he affirmed that screening hearsay on the basis of necessity and reliability had a fundamental constitutional aspect, because admitting unreliable hearsay endangered both the right to full answer and defence and, ultimately, the right to a fair trial. 28 He noted that while Parliament had occasionally chosen to create statutory hearsay exceptions applicable in criminal cases, when it did so it built in safeguards and protections that preserved the ability of the trial judge to ensure that it was reliable enough to put before the trier of fact. For example, the exception for business records requires proof that the records were kept and relied upon in the ordinary course of business and excludes records emanating from investigations in contemplation of legal proceedings, 29 while the exception for admitting a transcript of testimony from certain kinds of previous court proceedings rests on the accused having had the ability to fully cross-examine the earlier witness. 30 Turning to section 36, Ehrcke J reviewed the limited case law on the interpretation of section 36 and accepted that, despite the complex wording of the provision, the proper interpretation was that, 'although the section does not make compliant foreign records automatically admissible, it does have the effect of eliminating the need to establish the twin criteria of necessity and threshold reliability.' 31 In the end, this was its constitutional flaw. Justice Ehrcke noted that there were only two reliability 'protections' in section 36, and both were inadequate. First, the evidence must have been received in response to an MLAT request, which meant that a treaty was in place; this, he noted, 'will presumably eliminate the admission of records from manifestly corrupt and oppressive countries, since Canada would be unlikely to have concluded a treaty with such regimes.' 32 However, this provided little protection, since the kind of evidence sought to be introduced here could not be introduced by the government of Canada, either. 'Why,' Ehrcke J asked, 'should it be easier for the Crown to adduce foreign hearsay records than Canadian records?' Second, in section 36(2) the trier of fact was given discretion to consider the probative value of the evidence, which meant they could reject it if they found it to be unreliable. However, Ehrcke J opined, this was no substitute for scrutiny at the admissibility stage by the trial judgewhose role it is to keep unreliable hearsay from being placed before the trier of fact in the first place. 34 Accordingly, the provision was struck down as unconstitutional. Ehrcke J's findings were upheld on appeal by the British Columbia Court of Appeal. 35 The Court's judgment is somewhat revealing of the difficulty the Crown was having with the case, as it notes among other things the fact that the judgment under appeal had, in fact, been a result of the Crown's fourth attempt to adduce the documents, not all of which were even provided in English translation. The earlier attempts had featured recorded audio-video testimony by the officer from Thailand, which Ehrcke J had refused to put before the jury because of sound quality problems, 36 as well as the eventual refusal of the officer to come to Canada or testify via video link. 37 On appeal, the Crown had narrowed the range of the documents it sought to adduce, because it recognized the 'problematic' nature of many of the documents containing opinion and hearsay, and now only sought to adduce two printouts of travel history relating to two of the accused. However, it renewed its argument that hearsay received in response to an MLAT request did not have to meet the requirements of necessity and reliability, arguing on policy grounds that this 'does no more than "relax the rigid grip of Canada's hearsay law on the admissibility of foreign evidence."' 38 In a shifting of its argument in the court below, the Crown contended that the provision was still constitutional, because the trial judge retained the ability to exclude the evidence if its prejudicial effect outweighed its probative valueincluding any hearsay or opinion content. The Court began its analysis by noting that the provision had been part of the Act in its original form, which came into effect in 1988, a long time prior to when the principled approach to hearsay evidence had taken hold (and a point to which I will return below). Therefore, like Ehrcke J, the Court of Appeal agreed with the Crown's construction of the meaning of the provision: We agree with the Crown that s. 36 precludes a successful objection to the admissibility of a document solely on the basis that it contains hearsay or an opinion, which is a form of hearsay. We also agree with the Crown that, by reason of s. 36, a document containing hearsay can be tendered for the truth of its contents without the need to establish the evidence either falls within a recognized common law exception to the hearsay rule or meets the requirements of the principled approach to hearsay. To use the travel histories as an example, the Crown says because those documents were provided in response to a mutual legal assistance request they can, without more, be used to prove: (a) when the person whose name appears on the document entered or departed from Thailand; (b) the passport the person used; (c) the visa used (if any); 34 (d) where the entry or departure occurred (e.g., airport or checkpoint); and (e) in the case of air travel, the flight on which the person arrived or departed. 39 The issue was the provision's constitutionality, since it was clear from the Supreme Court of Canada's case law that hearsay could not be admitted via either a common law exception or a statutory exception unless those exceptions accounted for necessity and reliability. Shorn of those requirements, the provision endangered trial fairness. It was also no answer to argue, as the Crown had, that the trial judge could still exclude on the probative value versus prejudicial effect weighing, since that discretionary power only kicked in when the hearsay was found to be both necessary and reliable; it was designed to protect against situations when necessary and reliable hearsay might, for some reason particular to the case at hand, nonetheless affect trial fairness. 40 Accordingly, the Court of Appeal held that section 36 breached sections 7 and 11(d) of the Charter and therefore was of no force and effect. It did limit the scope of the judgment to 'evidence tendered by the Crown in a criminal trial,' and made 'no comment as to the application of section 36 in other contexts.' 41
THE CHICKENS CROSS PROVINCIAL BORDERS: RAJA At the time of writing, Rajatnaram has been fully applied only once, in the 2020 case of R v Raja which took place in the Canadian province of Ontario. 42 The battle was actually joined a year earlier, in R v Boyce, 43 a case that involved evidence obtained from Panama via an MLAT request. In that case, the Court of Appeal agreed with the finding of the British Columbia Court of Appeal in Rajatnaram that section 36(1) of the Act did indeed remove the trial judge's ability to screen MLAT-sourced hearsay for necessity and reliability. 44 However, the issue of whether this interpretation rendered the section unconstitutional had been raised for the first time on appeal, and as the matter had not been canvassed in the trial court the Court of Appeal declined to decide the issue. Raja arose from an alleged heroin trafficking operation where the drugs were being sent from Pakistan to addresses in Canada that were associated with the accused. Two of these shipments had been seized by Pakistani police, and Canada requested that Pakistan provide evidence regarding the seizures, via the mutual legal assistance provisions in the Vienna Narcotics Convention, 45 to which both states are parties. The Canadian request for assistance was quite detailed and specific, 46 but the information received in response was deficient in a number of ways, consisting of a letter from a government official, accompanied by unsworn and uncertified copies of documents, some of which were translated only in part. The Crown sought to rely on the documents for the sole purpose of demonstrating that the substance in the seized shipments had been heroin. 39 The accused made essentially the same argument that had been made in Rajatnaram, and with the same result. The trial judge, Wells J, noted that the Court of Appeal had already decided in Boyce that section 36(1) did remove necessity and reliability screening, and this was binding authority for this lower court. On the constitutional challenge, Justice Wells summarized the findings in Rajatnaram and agreed with themto admit hearsay of this sort without the opportunity to challenge it would undermine the accused's right to a fair trial and to full answer and defence, and thus breached sections 7 and 11(b) of the Charter. 47 Justice Wells also made some comments which threw cold water on the Crown's argument (from Rajaratnam) that some reliability attached to documents received via a request simply because they were the result of international cooperation: Here, the Crown seeks to rely on evidence that lacks even the most basic indicia of trustworthiness. Despite the details sought in the Request for Assistance, the Pakistan Documents are not certified to be true copies or accompanied by any oath or guarantee of truthfulness. If signatory countries to The Convention are truly committed to combating transnational crime, a commitment to effective assistance in prosecuting cases seems obvious. There was no evidence lead to suggest that the Pakistan authorities would be unwilling to testify via video-link for example. Were it the case that any country refused to make witnesses available to testify about documents provided under The Convention, this would call into serious question both their commitment to the cause, and the reliability of the evidence provided. Combating transnational crime, including global drug trafficking, is an important objective. In many instances, courts will need to be flexible in allowing for the presentation of evidence in different ways, particularly where that evidence is from another part of the world. However, that flexibility cannot extend so far as to allow for the admission of what would otherwise be inadmissible evidence, simply because it comes from another State pursuant to a treaty. It cannot be that officer notes from Pakistan in this case are somehow imbued with reliability (and therefore admissible for their truth), when notes of investigators from PRP could not be so tendered. In the result, the evidence was excluded. At the time of writing, there had been no appeal of the decision in Raja because the prosecution is still ongoing. 48 As a postscript, it may be worth noting the extent to which these decisions have and have not changed Canadian law on the subject. The British Columbia Court of Appeal is the highest court in the province of British Columbia, and Rajatnaram binds all trial courts of criminal jurisdiction in that province, which would include the British Columbia Provincial Court and the Supreme Court of British Columbia. The decision is not binding on courts in other provinces, but those courts are permitted to consider it and even adopt it, and the findings of senior appellate courts are often influential. This is in fact what happened in Boyce, as the Ontario Court of Appeal agreed with the British Columbia Court of Appeal's interpretation of section 36(1) in Rajatnaram. This makes two senior appellate courts that have agreed on that particular interpretation. As to the section's constitutional invalidity, Raja is from the Ontario Court of Justice, which is the lowest court of criminal jurisdiction in the province of Ontario and has no binding stare decisis-type authority. As a matter of judicial comity, other judges of that court are required to consider it but are free to depart from it if they disagree with its interpretation of the law. No other court in the province is bound to follow it. The Supreme Court of Canada, the findings of which are binding on all Canadian courts, has not yet considered the issue raised in these cases. Accordingly, the best way to describe the impact of these cases is to say that they have put Canadian law on the admissibility of MLATsourced hearsay in a state of flux (and have undoubtedly created headaches for the Crown and police).
ANALYSIS: LEVELLING THE TENSION BETWEEN SOVEREIGNTY AND COOPERATION It is important to note the actual effect of the case developments canvassed here. It is not the case that hearsay evidence that is received via MLAT is inadmissible at Canadian trials, even trials in British Columbia and Ontariorather, MLAT-sourced is put back on an equal footing with all the other evidence that might be led by the Crown in a criminal prosecution. That is to say, it must comply with the standard rules of evidence. While section 36 of the Act does cover opinion that might be contained in documents, the judgments do not seem to make any findings about this kind of evidence, and therefore, notionally, the section still applies to opinion. Practically, however, any opinion evidence that shows up will almost exclusively be hearsay in any event, since it will still amount to out of court statements offered to prove the truth of their contents, and thus will be required to meet the standard requirements for the admission of hearsay. Why is this a problem? And, more usefully for readers of this publication, is there anything useful or instructive about this case law, driven as it is by Canadian evidentiary and constitutional particulars? I submit that there is. The messiness in these cases may reflect some local specifics but is also an illustration of the well-known problem referred to earlier: the difficulty often encountered by requesting states (particularly common law states) in obtaining evidence from requested states in a form that is admissible in a prosecution. Introducing flexibility in this area has historically proven to be difficult. Criminal law, after all, is closely linked to the sovereignty of states as embodied in their legal and constitutional norms and traditions. International law contains various legal rules that are designed to respect and accommodate two realities: first, as I often tell my students (with slight comic effect), that there are over 190 different criminal law systems in the world, each of which thinks the others are 'doing it wrong;' and second, that states are motivated to 'protect' their territories and citizenry from foreign extensions of criminal law power. The international law of jurisdiction is instructive. For example, states are obliged to refrain from attempts to exercise prescriptive jurisdiction over an offence to which they have no reasonable connection. States are prohibited from exercising their enforcement powers on the territories of foreign states. At the same time, the system is not just reactive and protective but also actively respectful since the legal culture is one of mutual guarding of sovereignty. This can be seen in domestic laws of evidence; for example, states typically do not apply their own criminal procedure laws to the actions of foreign police, when those police were acting on their own territory and under the authority of their own state's law. 49 Evidence of both the conservative tendencies of states in criminal cooperation and the desire to temper those tendencies can be tracked at the international level. As far back as article 7(12) of the 1988 Vienna Convention we see a sovereignty safeguard common to MLATs: requests are to be 'executed in accordance with the domestic law of the requested Party;' yet the same article provides that 'to the extent not contrary to the domestic law of the requested Party and where possible,' the requesting state's wishes must be respected. 50 Identical language is found in article 18(17) of the UNTOC, 51 and article 46(17) of the UNCAC. 52 All of these reflect similar language in article 6 of the United Nations Model Treaty on Mutual Legal Assistance in Criminal Matters. 53 The simple idea is that if it is possible for the requested state to satisfy the evidentiary requirements of the requesting state, it should try to do so. On paper, at least, states often indicate that they are willing to do so. 54 There is also encouragement of requesting states to provide for flexibility in their domestic laws of evidence. 55 Regarding hearsay in particular, the Model Legislation on Mutual Legal Assistance in Criminal Matters published by the Commonwealth Secretariat rather gingerly suggests essentially the same approach as is found in the Canadian legislation, that in the law of the requesting state an MLAT-sourced document should not be inadmissible 'by reason only that it contains a statement which is hearsay.' 56 Less ambitiously, the 2000 UNDCP Model Foreign Evidence Bill proposes essentially an 'interests of justice' hearsay exception, where the trial court can take into account considerations such as whether the evidence is actually otherwise unavailable, its probative value, whether much would be accomplished by cross-examining the maker of the statement, and any prejudice to the defence. 57 State practice shows that there is some, albeit limited, receptivity to this latter idea among the common law states where this issue usually takes hold. 58 Similarly to the now-questioned Canadian approach, Ireland's law provides for automatic admission of the contents of an MLATsourced document 'as evidence of any fact stated in it of which oral evidence would be admissible.' 59 The United States uses this approach selectively (often via the MLAT itself, which is self-executing), 60 and has a formalized exception for MLAT-sourced business records, 61 though overall there is no uniform mechanism. 62 Australia's federal legislation begins by removing the application of the hearsay rule as a bar to admissibility, but supplements this with an 'interests of justice' discretion to exclude that resembles the approach in the UNDCP Model Bill. 63 South Africa's legislation appears to provide relaxed admissibility for statements taken after criminal proceedings have been instituted, but subjects hearsay gathered prior to the beginning of proceedings to its usual hearsay regime. 64 New Zealand, on the other hand, subjects MLATsourced hearsay to the normal rules of evidence. 65 As the current Canadian developments show, however, pushing domestic courts away from their entrenched legal standards regarding hearsay can be challenging. This reluctance is not necessarily a bad thing; the local law will often be grounded in good sense gained over decades or centuries of trial experience. Nor is it intuitive that the transnational nature of any particular case should mean compromising on trial fairness and/or disadvantaging the defence. This seems particularly apposite when we recall that the defence starts out from a point of relative disadvantage in virtually all criminal law systems, and moreover rarely has access to the foreign state in order to gather its own evidence or obtain means to challenge evidence that was gathered there. 66 The question, then, is this: is there a case for trial courts treating foreign-gathered evidence more flexibly than that gathered domestically? As noted above in Christurajah, Justice Ehrcke asked, 'Why should it be easier for the Crown to adduce foreign hearsay records than Canadian records?' 67 While the judge intended the question to be rhetorical, there is a policy-based answer, which is that successful international criminal cooperation may require it, at least in some form. The Commentary to the 2000 UNDCP Model Foreign Evidence Bill captures the point: The conditions of admissibility before a court for evidence obtained from other states will differ from state to state. Restrictive rules on admissibility will lead to such that is, the admission of the contents of documents for the truth of those contents. While it is beyond the scope of this article, both MLATs and implementing legislation often contain provisions that exempt from the hearsay rule material purporting to authenticate the documents which were the subject of the request. So, for example, a certificate signed by a foreign authority that certifies or explains the provenance of the documents is not treated as hearsay; the prosecution is not required to call as a witness a foreign official, simply to authenticate. Section 36 of the Canadian Act contains language to this effect. My overall impression is that this specific kind of relaxation of the hearsay rule tends not to be controversial. 59 evidence being excluded in many cases. The interests of justice are such that before evidence obtained from foreign states can be admissible, it must satisfy certain conditions, but it would seem contrary to those interests if it has to satisfy the rules of admissibility applicable to evidence obtained in the state itself; given the differences in requirements in different legal systems, this would unduly complicate and lengthen the judicial process. 68 Lurking in policy statements of this kind is an implicit criticism of the restrictive nature of evidence rules in common law states versus the broader approach in civil law states, which might raise hackles among the former. That said, a rational domestic system of evidence law, in principle, should be able to bend in some manner (consonant with national rules and standards) to accommodate the simple fact that foreign states do not do things in the same way as 'we' do, and in some cases cannot do them in that way. The differences in approach should not necessarily be fatal the integrity (and eventually the admissibility) of the evidence. This is what Justice Gerard La Forest tried to capture decades ago, in the extradition case of Canada v Schmidt, when he stated: I see nothing unjust in surrendering to a foreign country a person accused of having committed a crime there for trial in the ordinary way in accordance with the system for the administration of justice prevailing in that country simply because that system is substantially different from ours with different checks and balances. The judicial process in a foreign country must not be subjected to finicky evaluations against the rules governing the legal process in this country. 69 At the same time, however, flexibility should not come at the cost of procedural integrity and trial fairness. The finer point made by La Forest J was that the 'checks and balances' in domestic criminal law systems may look different from each other, but there must nonetheless be checks and balances. As reflected in the MLAT provisions from the suppression conventions canvassed above, it may be good policy for both requesting and requested states to be accommodating to foreign standards around evidence-gathering and admissibilitybut this should be accompanied by efforts on every side to keep the need for accommodation to a minimum.
CONCLUSION: PRACTICAL SOLUTIONS What can be done on a practical level? And, specific to this comment, what are potential solutions for a state like Canada where attempts to calibrate admission standards are scotched by the courts? One of the more obvious is precisely the one that was at issue here: the modification of the requesting state's evidentiary rules to accommodate the peculiarities of the foreign system. The Canadian legislation, originally passed in 1988, was an early example of this, though as discussed here, Canada's practice also shows that simply attempting to make foreign-sourced hearsay automatically admissible will sometimes be too crude a measure. There might be some middle ground between automatic admissibility and subjection to the state's ordinary hearsay rules, such as building presumptions (perhaps rebuttable) about the reliability of the evidence, or statutory direction to trial judges to take into account the international features of an investigation. The main point is that states interested in complying with the spirit of their obligations to facilitate cooperation should turn their minds to admissibility standards. The Canadian cases also offer a more prosaic point, which is that when states have access to MLAT-specific laws of evidence, it is important to keep them up to date. Rajatnaram happened, after all, because an experienced defence lawyer attacked the constitutionality of statutory wording created in 1988, using court authority from 2006. The argument was a clever one but could have been made much earlier; the Crown, for its part, did not foresee it and instead kept advancing an interpretation of the statute that it had been urging on the courts for decades, and ended up hoist with its own petard when that interpretation was found to be both correct and unconstitutional. The lesson, perhaps, is simply that legislation or other domestic laws meant to implement MLA practice need to be as modern as possible and keep pace with other criminal and constitutional law developments. One technological solution is increasingly obvious: having witnesses testify directly by way of video/internet connection, so as to prevent the need to adduce hearsay evidence in the first place. This is, in a sense, old hat because the technology required to do this has been around for some time, some MLATs already provide for it, 70 and many states currently avail themselves of it; Canada is one of these. 71 One of the silver linings of the COVID-19 pandemic is that much more extensive use has been made of virtual testimony, and both the technology and the overall skill level at using it have improved significantly in many jurisdictions. Article 11 of the new Second Additional Protocol on Enhanced Cooperation and Disclosure of Electronic Evidence to the Council of Europe's Convention on Cybercrime provides a thorough template for inter-state cooperation, 72 dealing with issues such as cost, translation, rules regarding privilege, and so on, that could provide guidance to states less familiar with the technique. 73 To be sure, this will not always be an available or effective solution; some states will lack the infrastructure or resources to provide witnesses in this way, and as happened in Rajatnaram itself the video and audio feed will not always be up to court standards. However, on the whole one suspects more extensive use could be made of this technique than is being done at the moment. At the very least, as Wells J suggested in Raja, the prosecution should be required to prove that they inquired into the possibility with the state from which the evidence came and provide some reason why it cannot be done. Wells J's reasons also suggest another practical point, albeit one that has been a preoccupation of MLAT practice for some time: for inter-state cooperation to work, the burden should not be placed entirely on requesting states; authorities in requested states must 'step up their game' and, at the very least, attempt to comply with the parameters of the request. The prosecution in Raja was hampered by the fact that the Pakistani authorities appear to have made only a half-hearted effort to provide the evidence in usable form. This led to Justice Wells' dry but poignant comment: 'If signatory countries to [the UNTOC] are truly committed to combating transnational crime, a commitment to effective assistance in prosecuting cases seems obvious.' 74 70 For example, the UNCAC (n 5) art 46(18). The review of implementation noted that many parties have handled such requests, and 20 of these stated they do so 'regularly or routinely'see Chrysikos (n 54) 473. 71 Canada Evidence Act, RSC 1985, c C-5, s 46(2). Indeed, in Lalji, above note 42, when the court ruled that the hearsay evidence would be excluded, the Crown simply opted to have the Australian witnesses testify "remotely," which suggests some form of video link . 72 ETS No 224 (2021). 73 ETS No 185 (2001). The text of Protocol and its explanatory report can be found online: <https://rm.coe.int/booklets-bc-2-protocols-guidance-notes-en-2022/1680a6992a> accessed 16 January 2023. 74 Raja (n 42) . In fairness, it is undoubtedly true that police and prosecutors the world over must deal with overstretched budgets and scarcity of time. Nonetheless, this case does tell us that the 'obligation' of accommodation first propounded globally in the Vienna Convention back in 1988 remains aspirational in some settings. A conclusory point: all of this might be made easier if there were more attention to transnational and comparative criminal law in the education and training of criminal justice officials (including defence lawyers) the world over. One recently-retired senior Canadian justice official has commented that, given the continuing expansion of transnational crime, 'lawyers and other professionals interested in criminal justice policy will need to be well versed in both domestic and comparative criminal law and procedure. They will be asked to translate international to domestic, and domestic to international.' 75 Operationally, it seems particularly important that (as the UNODC urged a decade ago), 'States make a concerted effort to carefully and fully explain the niceties of their laws to each other [and] make inquiries about the other country's legal systems whenever there is a doubt.' 76 This would make most sense in the context of heavilytrafficked bilateral or regional networks, at least for a start. 75 See generally Mirjan Damaska, 'Of Hearsay and its Analogues' (1992) 76 Minnesota Law Review 425. 10 See Bruce Zagaris, 'United States on Mutual Legal Assistance in Criminal Matters' in M Cherif Bassiouni, 6 Gail Kent, 'The Mutual Legal Assistance Problem Explained' (Centre for Internet Law & Society, 23 February 2015) <https://cyberlaw.stanford.edu/blog/2015/02/mutual-legal-assistance-problem-explained> accessed 16 January 2023. 7 Interesting and relevant examples are canvassed in UNODC, Manual on Mutual Legal Assistance and Extradition (2012) at 14. 8 ibid 16. 9 International Criminal Law, Volume II: Multilateral and Bilateral Enforcement Mechanisms (3rd edn, Nijhoff 2008) 385; Jeremy A Blumenthal, 'Shedding Some Light on Calls for Hearsay Reform: Civil Law Hearsay Rules in Historical and Modern Perspective' (2001) 13 Pace International Law Rev 93. 11 Markus Dubber, 'Comparative Criminal Law: Histories, Functions, Topics' (4 January 2018), 2-3, available SSRN <https://ssrn.com/abstract=3096540> accessed 16 January 2023. 12 Boister (n 2) 326-7.
13 I will not provide extensive citations to the relevant authorities or literature on Canadian hearsay law. The best general resources are: David M Paciocco, Palma Paciocco & Lee Steusser, The Law of Evidence (8th ed, Irwin (Thomson Reuters, 2019); and David Tanovich, Louis Strezos & the Hon S Casey Hill, McWilliams' Canadian Criminal Evidence (Looseleaf edn, Carswell, 2013) ch 7. 14 With its decision in R v Khan 2 SCR 531. Law, 2020) ch 4; Sidney N Lederman, Alan Bryant & Michelle Fuerst, Sopinka, Lederman & Bryant: The Law of Evidence (5th ed, LexisNexis Canada, 2018) ch 6; Peter Sankoff, The Law of Witnesses and Evidence in Canada
Criminal Justice (Mutual Assistance) Act 2008 (Ireland), s 73(8). 60 Michael Abbell, Obtaining Evidence Abroad in Criminal Cases 2010 (Nijhoff 2010). 61 18 US Code § 3505(a)(1). 62 See generally Christopher J Smith, Anthony Aminoff & Kelly Pearson, 'Gathering Gang Evidence Overseas' (2020) 68(5) US Attorneys Bulletin 47; Lauren Briggerman and others, 'Challenges to Obtaining Foreign Evidence in Cross-Border Criminal Cases' The Champion (November 2019) 30. 63 Foreign Evidence Act 1994, 1994/59, ss 24-5. 64 International Cooperation in Criminal Matters Act No 75 of 1996, ss 5(1) and 5(2). See Jamil Ddamulira Mujuzi, 'The South African International Cooperation in Criminal Matters Act and the Issue of Evidence' (2015) 48(2) De Jure 351, 375-8. 65 Mutual Assistance in Criminal Matters Act 1992, 1992/86, s 63(1). 66 Boister (n 2) 327-8. 67 Above n 25 and accompanying text.
Donald K Piragoff, KC, 'The Internationalization of Canadian Criminal Justice Policy' in Robert J Currie (ed), Transnational and Cross-Border Criminal Law: Canadian Perspectives (Irwin Law, forthcoming 2023). 76 UNODC (n 7) 16.
See Robert J Currie, 'The Evolution of the Law of Evidence: Plus Ça Change?' (2011) 15 Canadian Criminal Law Review 213.
Part I of the Constitution Act, 1982, being Schedule B to the Canada Act 1982 (UK), 1982, c 11.
R v Starr 2000 SCC 40 .
See, for example, R v Armour Pharmaceutical OJ No 5846 (Ontario Superior Court of Justice); R v Boyce 2019 ONCA 828 (Ontario Court of Appeal).
As far back as R v Corbett 1 SCR 670 and R v Potvin 1 SCR 525, the Supreme Court of Canada has maintained that the Charter mandates the continuation of this power.
2016 BCSC 2400 (Supreme Court of British Columbia), affirmed on appeal, R v Rajaratnam 2019 BCCA 209 (British Columbia Court of Appeal). The case name changed on appeal because one of the accused, Christurajah, was the subject of a separate procedural remedy, and his case became uncoupled from that of the other three. Accordingly, I will refer to the trial level decision as Christurajah and the appeal decision as Rajaratnam.
Specifically, they were charged with 'knowingly organizing, inducing, aiding, or abetting the coming into Canada of persons not in possession of a passport or other required travel documents' under s 117 of Canada's Immigration and Refugee Protection Act, SC 2001, c
ibid .
Personal communication from David Quayat, co-counsel for the Crown in Raja. Canadian criminal law does not allow for appeals of interlocutory decisions, and thus any appeal of this particular decision could only occur after the final trial verdict is rendered.
UNDCP Model Foreign Evidence Bill [Commentary] (2000) <https://www.unodc.org/documents/legaltools/model_foreign_evidence_bill__commentary.pdf> accessed 16 January 2023.
|
10.21203/rs.3.rs-1357212/v1
|
cc-by
| null | null |
openalex
|
Background: Global tropospheric ozone has doubled due to emissions from fossil fuel and biomass burning, and thus reduces plant primary productivity and crop yields. In contrast, little is known about how elevated tropospheric ozone affects soil microbial communities in the cropland ecosystem and whether such effects are sensitive to nitrogen (N) supply. Here, we examined the responses of bacterial and fungal communities in maize soils to elevated ozone (+60 ppb ozone) across different levels of N fertilization (+60, +120, and +240 kg N ha -1 yr -1 ). Results: Proteobacteria, Actinobacteria, Bacteroidetes, Chloro exi, Elusimicrobia, and Planctomycetes were altered by N fertilization or elevated ozone (P < 0.05). No interactive effects between N fertilization and elevated ozone were observed for bacterial and fungal communities (P > 0.1). While bacterial responses to N fertilization were phylogenetically conserved, bacterial and fungal responses to elevated ozone were phylogenetically conserved, showing universal homogeneous selection (homogeneous environmental conditions leading to more similar community structures). The ozone-responsive phyla were generally correlated (P < 0.05) with plant biomass, plant carbon (C) uptake, and soil dissolved organic C. Conclusions: Our study highlighted that microbial response to elevated ozone displayed a phylogenetic clustering pattern, suggesting that response strategies to elevated ozone stress may be a phylogenetically conserved ecological trait.Background Although nitrogen (N) fertilization improves crop production , its overuse has resulted in excessive N accumulation in soil, leading to higher N emissions from soil to the atmosphere . To date, tropospheric deposition of N in global ecosystems has increased and is projected to increase continuously within the rst half of the 21st century . Nitrogen fertilization in agricultural soils also contributes to nitrous oxide emissions, representing a signi cant greenhouse gas . Therefore, reducing N fertilization is necessary to mitigate global climate changes. Meanwhile, tropospheric ozone has increased since the industrial revolution because of the increased levels of reactive N oxide radicals and reduced volatile organic compounds [5, 6] . As an essential component of air pollution and greenhouse gas, many countries face elevated tropospheric ozone problem , with the maximum daily average ozone concentration at up to 70 ppb across China in 2018 and increasing 1-2% annually throughout the 21 st century [9, 10] . Tropospheric ozone can inhibit crop growth, photosynthesis, and owering , reducing crop production . In accordance, the elevated tropospheric ozone has decreased approximately 10% maize yields in the United States from 1980 to 2011 based on historical observations and reduced 6-8% annual crop yields in China . The N use e ciency of crops decreased by elevated tropospheric ozone through decreasing N uptake, which is one of the main reasons that drop crop yields . Both N fertilization and elevated tropospheric ozone might affect the soil microbial community, which plays critical roles in mediating geochemical cycles of C, N, phosphorus, and sulphur that support the plant growth . Long-term N fertilization decreased soil microbial biomass and diversity , and increased the ratio of Gram-positive bacteria to Gram-negative bacteria . In contrast, elevated ozone can either decrease or increase soil microbial diversity. The relative abundance of certain soil bacterial populations was altered by elevated ozone. For example, nitrifying bacteria and N-xing bacteria a liated to Sphingomonadaceae, Rhizobiaceae, and Nitrospiraceae were increased by elevated ozone in maize soils . In addition, soil nutrient availability and resource distribution were altered under elevated ozone due to alterations in the ratio of fungi to bacteria . Five-year elevated ozone treatment reduced soil organic C (5.6-17%) and N contents (8.2-27.8%), which reduced the N fertilization e ciency of crops . However, our previous studies found no interactive effects of N fertilization and elevated ozone on maize biomass and production . Whether there are interactive effects of N fertilization and elevated ozone on soil microbial communities remains obscure. A meta-analysis study suggests that microbial response to N fertilization is phylogenetically conserved , i.e., closely related microbial taxa respond more similarly to N fertilization than those that are distantly related. Microbial responses to other environmental changes, such as drought, extreme desiccation, and rewetting, are also phylogenetically conserved . In contrast, it is unclear whether microbial responses to elevated ozone are also phylogenetically conserved. . In this study, three N fertilization levels (60, 120, and 240 kg N ha -1 yr -1 ) and two ozone levels (ambient and ambient + 60 ppb ozone) were employed to investigate the effect of N fertilization levels and elevated ozone on soil microbial community during the whole growth cycle of maize. We aim to address the following questions: (i) whether and how interactions of N fertilization and elevated ozone alter soil microbial community; (ii) whether both soil microbial responses to N fertilization and elevated ozone are phylogenetically conserved in maize agro-ecosystem; (iii) which microbial populations are involved in response to N fertilization and elevated ozone, and whether there are similarity or difference between microbial populations that responded to N fertilization and elevated ozone.
Methods The study site and experimental manipulations The soils used in the present study were collected from an agricultural station located in Tangjiapu (40 (OTCs): N60, 60 kg N ha -1 yr -1 ; N120, 120 kg N ha -1 yr -1 ; and N240, 240 kg N ha -1 yr -1 . After the growth status of plants reached the 4-leave stage (4 maize leaves were grown), elevated ozone treatments were performed on June 30, 2019 to elevate the tropospheric ozone concentration by 60 ppb. Elevated ozone treatments were applied with an electrical discharge ozone generator (HY003, Chuangchen Co., Jinan, China), whose concentrations were monitored by an ultraviolet absorption ozone analyzer (Model 49i; Thermo Scienti c, Franklin, Massachusetts, USA). The OTCs supplied with ambient air were used as control. Six OTCs made by the toughened glass were used in this study; thus, there were three OTCs for elevated ozone treatments. In addition, three replicates for each N fertilization treatment were distributed in each OTC. In total, six treatments and nine replicates for each treatment in this study were conducted until the mature stage (September 24, 2019).
Sample collection Soil samples were collected at the end of September 2019. The soil samples were sieved (2 mm) to remove litter, roots, and stones. They were then put into airtight polypropylene bags, and placed in a cool box at 4C during transportation to the laboratory. Soil samples were then divided into two subsamples. Subsamples for soil geochemical analyses were stored at 4 C. Subsamples for microbial community composition analysis were stored at -80 C.
Determination of plant properties and soil geochemical properties Soil pH was measured with a pH meter (Model PHS-3C, Shanghai Precision and Scienti c Instrument Co. Ltd., Shanghai, China) after shaking the soil in deionized water (1:2.5 w/v) suspensions for 30 min. Soil total organic carbon (TOC) was determined by the potassium dichromate-volumetric method. Dissolved organic carbon (DOC) was extracted by adding 120 ml of deionized water to 40 g soil samples (1:3 w/v) as described . After being centrifuged at 4000 rpm for 10 min and passed through a 0.45 μm membrane, the ltered extracts were used for DOC analysis. Total N (TN) was determined by Kjeldahl digestion. Ammonium (NH 4 + ) and nitrate (NO 3 -) were extracted by 0.01 M potassium chloride (1:10 w/v) for 30 min and then detected by auto-analyzer (Alpkem, Perstorp Analytical Company, Wilsonville, OR, USA). Total phosphorus (TP) was measured using the molybdenum-antimony colorimetric method after being treated by melted sodium hydroxide. Available phosphorus (AP) was measured after being treated by hydrochloric acid-ammonium uoride extraction. Total potassium (TK) was determined by atomic absorption spectrophotometer (Z-2300, Hitachi, Japan) after being treated by melted sodium hydroxide, and available potassium (AK) was extracted by ammonium acetate. Plant biomass, plant carbon uptake, and plant N uptake were measured by an elemental analyzer (Vario EL III, Elementar, Germany) as described before . Illumina sequencing of 16S rRNA genes and ITS amplicons Following the instructions, the whole soil DNA was extracted from 0.5 g soil samples by PowerSoil Kit (MOBIO, Carlsbad, CA, USA). Bacterial 16S rRNA gene V4-V5 hypervariable regions were ampli ed using primers 515F (5'-GTGCCA GCM GCC GCG GTA A-3') and 907R (5'-CCG TCA ATT CCT TTG AGT TT-3') combined with adapter sequences and barcode sequences. The ITS2 regions were ampli ed using primers ITS3 (5'-GCA TCG ATG AAG AAC GCA GC-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). Puri ed amplicons were sequenced by Magigene Inc., Guangzhou, China, on a HiSeq2500 platform (Illumina Inc., San Diego, CA, USA). Chimera detection and removal were accomplished using the Gold Chimera-Free reference database via the USEARCH option in the UCHIME algorithm. Quality-ltered reads were truncated to an equal length. Unosie3 were applied to generate ASV table, and ASVs were calculated using the usearch -unoise3 command. The representative sequence of each ASV was assigned to a taxonomic lineage by classi ed against the SILVA database (version 132) for 16S rRNA gene and UNITE database (version 7.2) for ITS sequences.
Statistical analyses ANOVA was applied to test the effects of N fertilization and elevated ozone on plant and soil geochemical properties, microbial diversity. FDR correction was performed to adjust the P-values obtained for multiple comparisons. The alpha diversity indices were calculated using R functions 'alpha.g' in the 'vegan' and 'ieggr' packages. The nonmetric multidimensional scaling (NMDS) analysis based on the abundance weighted Bray-Curtis distance was used for comparing the microbial communities in different treatments. Permutational multivariate analysis of variance (Adonis) based on Bray-Curtis distance was used to determine the microbial differences between treatments . Canonical correlations analysis (CCA) was conducted to detect the interactions of plant properties, soil geochemical properties, and microbial communities . The function 'env t' in the 'vegan' package evaluated the association of microbial community variation and each environmental variable in CCA. The function 'permutest' in the 'vegan' package was used to test the signi cance of the CCA model. The signi cance of ASVs changed by elevated ozone or N fertilization was determined by the Student t-test. For N fertilization treatments, the signi cantly changed ASVs were rstly selected by P < 0.05 between N240 and N60. Then, ASVs that increased with N fertilization levels and ASVs that decreased with N fertilization levels were selected, respectively. For ozone treatments, the signi cantly changed ASVs were selected by P < 0.05 between ambient and elevated ozone treatments. The ASVs increased by N fertilization or elevated ozone treatments were de ned as positive responses. The ASVs decreased by N fertilization, or elevated ozone treatments were de ned as negative responses. Pearson correlation analysis was conducted to evaluate correlations between the signi cantly changed microbial populations with plant and soil geochemical properties. FDR correction was performed to adjust the P-values. To assess whether the microbial responses to N fertilization or elevated ozone are phylogenetically conserved, representative sequences of these signi cantly changed ASVs were aligned using the DECIPHER package . An ML tree with 100 bootstrap replications was constructed with RAxML v8.0, using the GTR + Gamma distribution model . We then applied consenTRAIT analysis to test whether an ASV's response to N fertilization or elevated ozone was related to the microbial phylogeny . The tree was traversed from the root to the tips, recording the deepest nodes where >90% of the descending tips (ASVs) shared the same directional response (a 'consensus' clade). The genetic depth (average distance of the node to its descending tips) and each consensus clade's size (total number of the descending tips) were calculated. The genetic depth of clades with a single descending tip (i.e., ASV) was calculated as half the branch length to the nearest neighbor as previously recommended . Finally, the mean genetic depth, τD, of the consensus clades sharing positive or negative responses was calculated. Simulated τD values were calculated by randomizing the responses among the tips 1000 times, to assess the statistical signi cance of phylogenetic conservation of N fertilization levels or elevated ozone. The probability of phylogenetic conservation (non-randomness) of the traits was calculated as the fraction of simulated τD values that were greater than or equal to the observed τD . The taxonomy of clades whose response to N fertilization levels or elevated ozone was signi cantly more positive or negative than expected by chance was calculated based on the number of ASVs that had a positive or negative response at each taxonomic level. We performed a two-tailed sher's exact test against each taxonomic level's equal distribution of positive and negative responses.
Results Plant and soil geochemical properties Both N fertilization and elevated ozone exhibited signi cant effects on most plant and soil geochemical properties (Table S1 ). Speci cally, N fertilization increased plant biomass, plant N uptake, plant C uptake, and soil ammonium (NH 4 + ) concentration, whereas decreased soil available phosphorus (AP) (P < 0.05, Table S2 ). In contrast, elevated ozone decreased plant biomass, plant C uptake, soil pH, dissolved organic carbon (DOC), and AP but increased soil NH 4 + and nitrate (NO 3 -). No interactive effects of N fertilization and elevated ozone were observed on plant and soil geochemical properties except for soil total potassium (TK) and available potassium (AK) (P < 0.05, Table S1 ).
Microbial community composition and diversity After resampling at 45 000 reads per sample, a total of 2 430 000 amplicon sequences for 16S rRNA gene representing bacterial communities were generated, resulting in 18 260 amplicon sequence variants (ASVs). Most of these sequences were a liated to Proteobacteria (24.8-26.3%), Acidobacteria (15.6-17.7%), and Chloro exi (12.7-14.6%) (Fig. S1a ). The most abundant genera included Anaerolineaceae UTCFX1 (3.1-3.8%), Nitrosomonadaceae MND1 (1.8-2.5%), and Pyrinomonadaceae RB41 (1.9-2.3%) (Fig. S1b ). Bacterial alpha diversity (Shannon index) was decreased (P < 0.05) by N fertilization (Fig. 1a ) but was not affected by elevated ozone (Fig. 1b ). No interactive effect between N fertilization and elevated ozone was observed on bacterial alpha diversity (Table S3 ). Similarly, a total of 1 684 152 ITS sequences were generated after resampling at 31 188 reads per sample, resulting in 4 036 ASVs representing fungal communities. Most sequences were a liated to Ascomycota (42.3-48.8%), Glomeromycota (9.6-16.8%), and Basidiomycota (3.6-6.4%) (Fig. S1c ). The most abundant genera included Gibberella (6.0-9.4%), Claroideoglomus (4.1-6.4%), and Fusarium (4.0-5.0%) (Fig. S1d ). Contrary to bacteria, fungal alpha diversity was not affected by N fertilization (Fig. 1a ) but was decreased (P < 0.05) by elevated ozone (Fig. 1b ). No interactive effect between N fertilization and elevated ozone was observed on fungal alpha diversity (Table S3 ). There were signi cant (Adonis, P < 0.05) main effects of N fertilization and elevated ozone on both bacterial and fungal communities (Table S4 ). The effects of N fertilization (F = 1.58) and elevated ozone (F = 1.66) on the bacterial community were generally equal, but the effect of elevated ozone on the fungal community (F = 2.09) was much larger than that of N fertilization (F = 1.36) (Fig. 1c ). There was no interaction between N fertilization and elevated ozone on microbial communities (P > 0.1, Table S4 ).
Phylogenetic conservation of microbial responses to N fertilization A total of 1 593 bacterial ASVs were changed in relative abundance by N fertilization (P < 0.05, Fig. 2a ). These ASVs were mainly a liated to Proteobacteria (31.3%), Planctomycetes (10.5%), Chloro exi (10.4%), and Bacteroidetes (10.2%). 26.0% of the signi cantly changed ASVs were increased relative abundances by at least two-fold by N fertilization, and 45.3% of the signi cantly changed ASVs were decreased by at least 50% by N fertilization (Fig. S2a ). 86.2% of the ASVs a liated with Actinobacteria was increased by N fertilization (two-tailed exact test, P < 0.05, Fig. 2c ). On the contrary, most of the ASVs a liated to Gammaproteobacteria (69.2%), Deltaproteobacteria (75.5%), Bacteroidetes (69.3%), Elusimicrobia (91.3%), and Planctomycetes (65.5%) were decreased by N fertilization, suggesting that bacterial response to N fertilization was largely at the phylum level. We also examined other taxonomic levels and found a consistent response to N fertilization (Fig. 2c ). The mean genetic depth (τD) of ASVs with both positive and negative responses ranged from 0.041 to 0.049 (average τD = 0.045, permutation test, P < 0.05, Table 1 ), demonstrating around 9% of average sequence dissimilarity in the 16S rRNA gene amplicon showed consistent response to N fertilization. A total of 96 fungal ASVs were changed in relative abundance by N fertilization, which was mainly a liated to Ascomycota (26.0%) and Glomeromycota (20.8%) (Fig. 2b ). Even though 64% (16 out of 25 ASVs) of the ASVs a liated to Ascomycota and 80% (16 out of 20 ASVs) of the ASVs a liated to Glomeromycota were decreased by N fertilization (Fig. S2a ), the changes were not signi cant (two-tailed exact test, P > 0.05, Fig. 2c ). Besides, The τD of ASVs with both positive and negative responses were not signi cant (permutation test, P > 0.05, Table 1 ), indicating that fungal responses to N fertilization were not phylogenetic conserved.
Phylogenetic conservation of microbial responses to elevated ozone A total of 1 387 bacterial ASVs were changed by elevated ozone (Fig. 3a ). 25.6% (355 out of 1 387 ASVs) of these ASVs were increased in relative abundances by at least two folds by elevated ozone, and 29.0% (402 out of 1 387 ASVs) of these ASVs were decreased at least 50% by elevated ozone. Most of the ASVs a liated to Alphaproteobacteria (71.6%), Actinobacteria (69.4%), and Chloro exi (81.6%) were increased, whereas most of the ASVs a liated to Gammaproteobacteria (68.1%), Bacteroidetes (90.1%), and Elusimicrobia (88.9%) were decreased by elevated ozone (two-tailed exact test, P < 0.05, Fig. S2b ). , These suggested that bacterial responses to elevated ozone occurred at the phylum level. We also examined other taxonomic levels and found a consistent response to elevated ozone (Fig. 3c ). The mean τD of ASVs with positive and negative responses ranged from 0.050 to 0.052 (average τD = 0.051, permutation test, P < 0.05, Table 1 ), demonstrating around 10.2% of average sequence dissimilarity in the 16S rRNA gene amplicon showed consistent response to elevated ozone. A total of 220 fungal ASVs were signi cantly changed by elevated ozone (Fig. 3b ). Among them, 33.6% of these ASVs were increased in relative abundances by at least two folds by elevated ozone, and 45.9% of these ASVs were decreased by at least 50% (Fig. S2b ). These 220 fungal ASVs were mainly a liated to Ascomycota (27.3%) and Glomeromycota (25.9%). 71.7% (43 out of 60 ASVs) of the ASVs a liated to Ascomycota were increased, whereas 93.0% (53 out of 57 ASVs ) of the ASVs a liated to Glomeromycota were decreased by elevated ozone (two-tailed exact test, P < 0.05, Fig. S2b ). All taxonomic levels showed consistent responses to elevated ozone (Fig. 3c ). The mean τD of ASVs with positive and negative responses ranged from 0.18 to 0.26 (average τD = 0.22, permutation test, P < 0.05, Table 1 ). Comparison of microbial responses to N fertilization and elevated ozone Phylum Proteobacteria, Actinobacteria, and Bacteroidetes were signi cantly changed by both N fertilization and elevated ozone (Figs. 2 and 3 ). However, only 3% of bacterial ASVs and 2% of fungal ASVs were overlapped in response to N fertilization and elevated ozone (Fig. S3 ), suggesting that N fertilization and elevated ozone changed different microbial populations. The average τD of bacterial response to N fertilization was 0.045, suggesting that bacterial response to N fertilization was between family and genus levels (Fig. S4 ). However, the average τD of bacterial response to elevated ozone was 0.051, suggesting a bacterial response to N fertilization was between family and order levels. Hence, bacterial response to elevated ozone was slightly more deeply conserved than to N fertilization. Relationships of the signi cantly changed microbial populations with plant and soil geochemical properties Plant biomass, plant N uptake, and plant C uptake were positively correlated with Actinobacteria, which were increased by N fertilization (P < 0.05, Table 2 ). In contrast, those properties were negatively correlated with Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Elusimicrobia, and Planctomycetes, all of which were decreased by N fertilization. Plant biomass and plant C uptake were also negatively correlated with the phylum Alphaproteobacteria and Chloro exi (P < 0.05, Table 3 ), both of which were increased by elevated ozone. However, they were positively correlated with all phyla that showed negative responses to elevated ozone. Soil DOC was positively correlated with bacterial phyla that showed positive responses to elevated ozone but was negatively correlated with Alphaproteobacteria, which was increased by elevated ozone. For fungal communities, plant biomass and plant C uptake were marginally and positively correlated (P < 0.1) with Glomeromycota, showing a negative response to elevated ozone. Plant biomass and plant C uptake were negatively correlated (P < 0.05) with Ascomycota, positively responding to elevated ozone.
Discussion Long-term N fertilization usually decreased both bacterial and fungal alpha diversity [29, 30] , especially under high N fertilization levels . In this study, N fertilization also decreased (P < 0.05) bacterial alpha diversity (Fig. 1 ). In contrast, no signi cant effect of N fertilization on fungal alpha diversity was observed, despite a decreasing trend of fungal alpha diversity with increasing N fertilization levels. This may be due to the shorter duration of N fertilization than other studies . Elevated ozone decreased (P < 0.05) fungal alpha diversity, which was also found in soils of two endemic trees in subtropical China . No interactive effects of N fertilization and elevated ozone were observed on plants and most soil geochemical properties closely correlated with microbial community , indicating that N fertilization could not alleviate the adverse effect of elevated ozone on plant and microbial communities. Together, our ndings revealed no interactive effect of N fertilization and elevated ozone at the tested levels on soil microbial community. Microbial responses to environmental changes can be phylogenetic conserved, including drought, speci c carbon resources utilization, precipitation, and N fertilization [20, 22, 23, 28] . In this study, bacteria with positive and negative responses to N fertilization were phylogenetically conserved at a genetic depth (τD) of 0.041 and 0.049 (Table 1 ). A previous study showed that the positive response of bacteria to over three years' N addition was conserved at a genetic depth of 0.020 . A meta-analysis of soil bacterial communities from 13 eld experiments across ve continents was conducted. Results showed that bacterial responses to N addition were phylogenetically conserved within (τD = 0.018) and across sites (τD = 0.017) , which are comparable but smaller than our observation (τD = 0.045), probably owing to the larger more signi cant amount of N used in this study. Bacterial responses to elevated ozone were also phylogenetically conserved. Bacterial positive and negative responses to elevated ozone were phylogenetically conserved at a genetic depth (τD) of 0.051 and 0.052, which are slightly higher than the conserved genetic depths in response to N fertilization. Similarly, bacterial responses to drought and nitrogen fertilization were also conserved in similar phylogenetic depth based on eld manipulation of precipitation and nitrogen fertilization for over three years . The fungal community was more in uenced by elevated ozone than N fertilization (Fig. 1 ), with more prominent τD value in response to elevated ozone. These results indicated that the fungal responses to elevated ozone are more phylogenetically conserved than N fertilization. Similarly, the response of fungal community to drought was more conserved than that to N fertilization . Hence, microbial stress response might be more conserved than resource utilization , as reactions to stress may involve the interactions of many parts of the genome , more than what is required for resource utilization. Nitrogen fertilization increased the relative abundance of Actinobacteria, but decreased the relative abundance of Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Elusimicrobia, and Planctomycetes (Fig. 2 ). The increase of Actinobacteria by N fertilization was consistent with a previous study showing that Actinobacteria was increased by N addition . The rise of Actinobacteria by N fertilization was highly correlated with the increase of plant biomass and soil NH 4 + . This is consistent with a long-term N fertilization study showing that N fertilization promoted the growth of Actinobacteria in agro-ecosystems, coinciding with increased soil available N by N fertilization . The phylogenetically conserved phyla showing negative responses to N fertilization were Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Elusimicrobia, and Planctomycetes, also broadly consistent with a previous study . Some bacterial populations a liated with Proteobacteria and Planctomycetes are N-xing populations , dispensable upon N fertilization. Elevated ozone increased the relative abundance of Alphaproteobacteria, Actinobacteria, and Chloro exi, but decreased Gammaproteobacteria, Bacteroidetes, and Elusimicrobia (Fig. 3 ). Chloro exi, increased by elevated ozone in this study, was also found as one of the dominant bacterial phyla after treatment with ozone in the bioreactor , suggesting strong resistance of Chloro exi to ozone. The increase of soil NO 3 -by elevated ozone can be attributed to the increase of nitrite-oxidizing Chloro exi , thus favoring nitrate-reducing Chloro exi . Actinobacteria was increased by elevated ozone. The increase of Actinobacteria was mainly correlated with the increase of soil NH 4 + and NO 3 -, which provide more available N for the growth of Actinobacteria . Bacteroidetes was decreased by elevated ozone in this study. Bacteroidetes utilizes complex algal and plant-derived polysaccharides as carbon and energy resources . Hence, the decrease of plant biomass and soil DOC by elevated ozone might induce the decrease of Bacteroidetes, consistent with our observations of positive correlation (P < 0.05) between plant biomass, soil DOC and Bacteroidetes (Table 3 ). The relative abundance of Actinobacteria was increased, while those of Gammaproteobacteria, Bacteroidetes, and Elusimicrobia were decreased by both N fertilization and elevated ozone. However, only 3% of bacterial ASVs were changed by N fertilization and elevated ozone, indicating that N fertilization and elevated ozone in uenced different bacterial populations at lower taxonomic levels. Only fungal response to elevated ozone was phylogenetically conserved (Table 1 ). Elevated ozone increased the relative abundance of Ascomycota, but decreased the relative abundance of Glomeromycota (Fig. 3 ). Plant properties and soil DOC showed signi cant correlations with fungal populations changed by elevated ozone (Table 3 ). Ascomycota, one of the major components of plant pathogenic fungi , presented a negative correlation (P < 0.001) with plant properties, suggesting a potential threat to plant health under increasing ozone. Glomeromycota, dominated by arbuscular mycorrhizal fungi (AMF), showed a negative response to elevated ozone. Recent study also found that elevated ozone changed AMF community composition and decreased AMF colonization . The growth of Glomeromycota depends on soil DOC, also mainly derived from root exudation of plant . A recent analysis including 239 studies exploring dry root mass of woody plants found that elevated ozone generally decreased root biomass . Hence, elevated ozone reduces typically the allocation of plant C resources to the soil, consistent with the reduced soil DOC by elevated ozone in this study. Glomeromycota forms symbioses with the roots and contribute to plant growth and production [43, 44] , and enhance environmental adaption of the plant to complicated environment . The inoculation of AMF increased 68% of shoot biomass and 131% of root biomass when the ozone concentrations were over 80 ppb, and then increased crop production under elevated ozone stress based on a meta-analysis including 20 studies . Therefore, the increase of Ascomycota and the decrease of Glomeromycota under elevated ozone might result in stunted growth and plant disease and further decrease plant biomass and production.
Conclusions In summary, we provide evidence that microbial responses to elevated ozone are phylogenetically conserved. As no interactive effect of N fertilization and elevated ozone on microbial community and plant and soil geochemical properties, the adverse effect of elevated ozone on plant and microbial community could not be alleviated by N fertilization. Furthermore, the decrease of AMF induced by elevated ozone would aggravate the adverse effect on crops if no policy were proposed to retard the increase of tropospheric ozone. Moreover, the decrease of speci c microbial species may result in the extinction of microbial species, warranting attention to protecting microbial diversity and agriculture development from the damage of increasing tropospheric ozone.
Declarations Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable. Tables Figures Figures
Figure 2 Phylogenetic 2 Figure 2 Phylogenetic distribution of the ASVs changed by N fertilization. Phylogenetic tree of bacterial ASVs (a) and fungal ASVs (b) that changed by N fertilization. Taxonomic levels of microbial response to N fertilization (c). The response signi cantly more positive (red) or negative (blue) or no signi cance (black) than expected by chance (two-tailed exact test; P < 0.05) are shown. Only the phylum contains >10 bacterial ASVs are shown. The percent of ASVs that were increased (red) or decreased (blue) by N fertilization are plotted in the bar graph to the right. Higher taxonomic levels are listed in black (e.g., the class Alphaproteobacteria) when only the lower levels are signi cant.
Table 1 1 Mean genetic depth (τD) of consensus clades as calculated with the consenTRAIT algorithm.Bold indicates that the response is signi cantly associated with phylogeny (permutation test; P <0.05). Consensus clades are the phylogenetic clades in which >90% of the descendant ASVs show the same direction of response. Bacteria Fungi Positive response Negative response Positive response Negative response N fertilization 0.041 0.049 0.101 0.135 Elevated ozone 0.051 0.052 0.255
Table 2 2 Pearson's correlation of plant and soil geochemical properties with ASVs responded consistently to N fertilization at phylum level. Actinobacteria Gammaproteobacteria Deltaproteobacteria Bacteroidetes Elusimicrobia Planctomycetes r P r P r P r P r P r P Plant biomass 0.54 <0.001 -0.55 -0.59 <0.001 -0.39 0.016 -0.37 -0.59 <0.001 Plant N uptake 0.67 <0.001 -0.66 <0.001 -0.70 <0.001 -0.58 <0.001 -0.57 <0.001 -0.72 <0.001 Plant C uptake 0.56 <0.001 -0.56 <0.001 -0.60 <0.001 -0.39 0.016 -0.37 -0.58 <0.001 pH -0.05 0.832 0.13 0.590 0.02 0.922 0.09 0.780 0.14 0.538 -0.02 0.900 TOC 0.22 0.234 -0.18 0.404 -0.09 0.780 0.07 0.782 -0.03 0.900 0.00 0.975 TN 0.06 0.808 -0.03 0.900 0.03 0.900 -0.03 0.900 0.01 0.952 -0.04 0.900 TP 0.24 0.201 -0.05 0.832 0.02 0.900 0.15 0.516 0.09 0.780 0.06 0.832 TK -0.12 0.644 0.07 0.782 0.15 0.517 0.26 0.134 0.16 0.479 0.17 0.440 DOC 0.25 0.164 0.03 0.900 0.10 0.780 0.21 0.295 0.36 0.029 0.08 0.782 NH 4 + 0.29 0.098 -0.31 0.071 -0.26 0.134 -0.22 0.234 -0.27 0.134 -0.29 0.098 NO 3 - 0.14 0.553 -0.09 0.782 -0.07 0.782 -0.08 0.782 -0.17 0.461 -0.06 0.832 AP -0.27 0.134 0.28 0.114 0.33 0.054 0.32 0.065 0.41 0.012 0.36 0.028 0.08 0.782 -0.05 0.833 -0.06 0.808 -0.11 0.683 -0.08 0.782 -0.11 0.685 Bold values represent P < 0.05 after adjusted by FDR.
Table 3 3 Pearson's correlation of plant and soil geochemical properties with ASVs responded consistently to elevated ozone at phylum level. Alphaproteobacteria Actinobacteria Chloro exi Gammaproteobacteria Bacteroidetes Elusimicrobia Ascomycota Glome r P r P r P r P r P r P r P r Plant -0.44 0.010 -0.24 0.194 -0.48 0.004 0.64 <0.001 0.35 0.046 0.40 0.017 -0.60 <0.001 0.32 biomass Plant N 0.03 0.896 0.10 0.650 -0.09 0.699 0.28 0.112 -0.01 0.942 -0.08 0.714 -0.30 0.085 0.02 uptake Plant C -0.43 0.010 -0.22 0.244 -0.47 0.004 0.63 <0.001 0.35 0.046 0.41 0.015 -0.59 <0.001 0.33 uptake pH -0.32 0.069 -0.35 0.046 -0.27 0.133 0.41 0.017 0.22 0.244 0.22 0.242 -0.33 0.059 0.19 TOC 0.09 0.713 0.13 0.542 -0.04 0.882 0.00 0.987 0.07 0.749 0.04 0.882 -0.03 0.896 0.08 TN 0.08 0.714 0.08 0.714 0.15 0.478 0.14 0.510 0.04 0.882 0.15 0.463 -0.05 0.821 -0.03 TP -0.10 0.641 0.02 0.914 -0.16 0.463 0.15 0.463 0.20 0.297 0.24 0.192 -0.01 0.945 0.15 TK -0.12 0.568 -0.09 0.705 -0.12 0.568 0.02 0.942 0.19 0.353 0.08 0.713 -0.13 0.542 0.19 DOC -0.36 0.042 -0.11 0.593 -0.24 0.196 0.42 0.014 0.35 0.046 0.44 0.010 -0.29 0.101 0.47 NH 4 + 0.31 0.082 0.29 0.094 0.21 0.266 -0.14 0.510 -0.13 0.542 -0.12 0.584 0.26 0.140 -0.15 NO 3 - 0.44 0.010 0.27 0.133 0.33 0.059 -0.37 0.040 -0.35 0.046 -0.32 0.069 0.31 0.082 -0.13 AP -0.21 0.266 -0.26 0.155 -0.18 0.367 0.16 0.435 0.27 0.136 0.37 0.040 -0.08 0.714 0.30 AK -0.08 0.713 0.07 0.738 0.07 0.714 0.06 0.802 -0.01 0.942 0.18 0.366 0.17 0.424 0.02 Bold values represent P < 0.05 after adjusted by FDR.
|
10.1007/s12021-020-09481-9
|
cc-by
| null | null |
openalex
|
Human brain atlases have been evolving tremendously, propelled recently by brain big projects, and driven by sophisticated imaging techniques, advanced brain mapping methods, vast data, analytical strategies, and powerful computing. We overview here this evolution in four categories: content, applications, functionality, and availability, in contrast to other works limited mostly to content. Four atlas generations are distinguished: early cortical maps, print stereotactic atlases, early digital atlases, and advanced brain atlas platforms, and 5 avenues in electronic atlases spanning the last two generations. Content-wise, new electronic atlases are categorized into eight groups considering their scope, parcellation, modality, plurality, scale, ethnicity, abnormality, and a mixture of them. Atlas content developments in these groups are heading in 23 various directions. Application-wise, we overview atlases in neuroeducation, research, and clinics, including stereotactic and functional neurosurgery, neuroradiology, neurology, and stroke. Functionality-wise, tools and functionalities are addressed for atlas creation, navigation, individualization, enabling operations, and application-specific. Availability is discussed in media and platforms, ranging from mobile solutions to leading-edge supercomputers, with three accessibility levels. The major application-wise shift has been from research to clinical practice, particularly in stereotactic and functional neurosurgery, although clinical applications are still lagging behind the atlas content progress. Atlas functionality also has been relatively neglected until recently, as the management of brain data explosion requires powerful tools. We suggest that the future human brain atlas-related research and development activities shall be founded on and benefit from a standard framework containing the core virtual brain model cum the brain atlas platform general architecture. Keywords Human brain atlas . Brain atlas evolution . Brain atlas generations . Brain atlas review . Brain atlas platforms * Wieslaw L. NowinskiIntroduction We witness in recent years a tremendous explosion of human brain atlas projects with various goals, scopes, and sizes, as addressed, for instance, in (Amunts et al. 2014; Frackowiak and Markram 2015; Nowinski 2017a; Hess et al. 2018 ). This explosion is propelled by brain-related big and well-funded initiatives and projects, including The BRAIN Initiative (Brain Research through Advancing Innovate Neurotechnologies) (BRAIN Working Group 2014) to develop technology to catalyze neuroscience discovery (Jorgenson et al. 2015) ; The Human Brain Project to create a research infrastructure to decode the human brain, reconstruct the brain's multiscale organization, and create brain-inspired information technology (Amunts et al. 2016) ; The Human Connectome Project to map structural and functional connections in the brain in order to study the relationship between brain circuits and behavior (Van Essen et al. 2013) ; The Allen Brain Atlas to map gene expression (Sunkin et al. 2013) ; The Big Brain to obtain ultrahigh resolution neuroimages (Amunts et al. 2013) ; The Blue Brain Project to simulate neocortical micro-circuitry (Markram et al. 2015) ; The CONNECT project to combine macro-and micro-structure (Assaf et al. 2013) ; the Brainnetome project to understand the brain and its disorders, develop methods of brain network analysis at different scales, and create the brainnetome atlas (Jiang 2013) ; the Chinese Color Nest Project to study human connectomics across the life span (Zuo et al. 2017) ; and the Japanese Brain/MINDS (Brain Mapping by Integrating Neurotechnologies for Disease Studies) project to further understand the human brain and neuropsychiatric disorders through ''translatable'' biomarkers (Sadato et al. 2019) . Therefore, with the new acquisition techniques introduced and big data acquired, sophisticated applications and tools developed, and novel concepts proposed, this explosion dynamically changes over time the concept, role, and understanding of a human brain atlas. Consequently, it is believed that the use of big digital science to neuroscience will create new avenues for the development of a modern human cerebral cartography (Frackowiak and Markram 2015) . Two major forces driving this brain atlas development these are human curiosity along with scientificbased interest empowered by the developments in brain mapping technology and computing, and clinical needs urged by the growing cost of brain disorders and society aging. The purpose of this state-of-the-art review is to attempt capturing the evolution of human brain atlases as well as to demonstrate the immense breadth of the ongoing work and its tremendous potential. We track this process of evolution over time, identify its numerous directions and categorize them, try to distinguish brain atlas generations, and capture the present state. Typically this evolution is considered in terms of atlas content, particularly, in research applications. However, the usefulness of human brain atlases depends not only on the atlas content but also on the functionality enabling and supporting various atlas-based applications as well as atlas availability. Therefore, our goal here is to take a wider perspective and address the human brain atlas development in terms of four major categories: (1) atlas content; (2) atlas applications in various areas; (3) functionality enabling and facilitating atlas use; and (4) availability in terms of access, media, and platforms enabling the atlas delivery to its user. The rest of the paper is organized as follows. The brain atlas evolution is reviewed in Sect. 2 in terms of atlas content (categorized into 8 main groups from 23 directions for the new electronic brain atlases taking into account diverse criteria), applications, functionality, and availability. In Sect. 3 four generations of brain atlases are distinguished and the process of atlas evolution is captured diagrammatically, followed in Sect. 4 by the discussion along with some suggested future directions.
Evolution of Human Brain Atlases We track below the evolution of human brain atlases in terms of content, applications, functionality, and availability.
Evolution of Brain Atlas Content The brain atlas content is the richest and most dynamic category whose development proceeds along multiple divisions, including postmortem versus in vivo data, whole brain versus specific cerebral regions, structure versus function, single data acquisition modality versus multi-modal data, single brain specimen and individual features versus a population of specimens and/or aspects, in health versus diseased, static print versus dynamic digital, single atlas versus multi-atlases, slow versus fast dynamic, and mono scale versus multi-scale, among others. The initial development of cerebral cortical maps was carried out predominantly in a single direction, meaning studying the cortical parcellation. Several early maps of the parcellated cerebral cortex were created in the first three decades of the 20th century by Campbell (1905) , Brodmann (1909) , Vogt and Vogt (1919) , Flechsig (1920) , and Von Economo and Koskinas (1925) . These first, postmortem, hand-drawn cortical maps were produced for a single modality, cytoarchitectonics (Brodmann 1909; Von Economo and Koskinas 1925) or myeloarchitectonics (Vogt and Vogt 1919; Flechsig 1920) , and they varied in terms of the number of the parcellated cortical areas. Namely, in the neocortex Campbell (1905) identified 14 areas, Brodmann (1909) 44 areas, Von Economo and Koskinas (1925) 54 areas, and Vogt and Vogt (1919) 185 areas. This process of cortical parcellation pioneered by Brodmann and the other early brain mappers a century ago continues until the present time being extended (1) from schematic two-dimensional (2D) single brain-derived surface drawings to multi-modal, populationbased probabilistic three-dimensional (3D) maps (Glasser et al. 2016 ) facilitating to study intersubject variability; and (2) from pure visual inspection of the examined material to the application of robust, objective and observer-independent cortical parcellation rules based on quantitative criteria and statistical measures (Amunts and Zilles 2015) , additionally enhanced by employing in vivo mapping with high-field magnetic resonance imaging (Geyer et al. 2011) . The need in neurosurgery to localize cerebral structures in the pre-tomographic imaging era caused the creation of stereotactic brain atlases (a review of print and electronic stereotactic atlases of the human brain is presented by Alho et al., (2011) ). These initially print atlases represented a big step forward in atlas development both in terms of atlas content and concept. In the 1950th stereotactic brain atlases were produced by Speigel and Wycis (1952) , Talairach et al. (1957) , and Schaltenbrand and Bailey (1959) . This development was continued by Andrew and Watkins (1969) , Van Buren and Borke (1972) , Schaltenbrand and Wahren (1977) , Afshar et al. (1978 ), and Talairach and Tournoux (1988 , 1993) . The major content-wise progress was made in four main directions: (1) from a few maps capturing the state-of-the-art about the brain to brain atlases applicable clinically; (2) from cerebral cortical maps to atlases of the whole brain (or its specific parts, including subcortical structures, cerebellum, and brainstem); (3) from a single specimen to multiple specimens with marked anatomic variability, although without any probabilistic maps yet (e.g., Schaltenbrand and Wahren (1977) used 111 brain specimens to create their atlas); and (4) integrating structure with function (neuroelectrophysiologic stimulation) like in the Schaltenbrand and Wahren atlas. Besides stereotactic, some other print atlases were published for neuroradiology, neurosurgery, neuroscience, and medical education and training, among others, a brain atlas for computed tomography (Takayoshi and Hirano 1978) , an atlas of the hippocampus (Duvernoy 1988) , an atlas of brain function (Orrison 1995) , an atlas of the brain stem and cerebellum with surface anatomy and vascularization (Duvernoy 1995) , an atlas of morphology and functional neuroanatomy (Scarabino et al. 2006) , an atlas of the brain stem and cerebellum with 9.4T images of 40-60 micron resolution (Naidich et al. 2009) , and the Netter's atlas of neuroscience (Felten et al, 2015) . In particular, some stereo brain atlases are created as a 3D depth perception is essential in neurosurgical routine. Bassett (1952) produced a stereoscopic atlas of human anatomy with stereo cadaveric images of the central nervous system, head, and neck. Poletti (1985) built a stereo atlas of operative microneurosurgery with stereo photographs taken intraoperatively. Kraus and Bailey (1994) created a stereo atlas of microsurgical neuroanatomy with successive surgical steps recorded photographically; moreover, a binocular viewer is attached to the atlas to perceive depth. A natural step forward in brain atlasing was the development of computerized electronic brain atlases aiming to overcome limitations of their print counterparts, such as static content, image plate sparseness, lack or limited functionality, cumbersome use, lack of interactivity, and difficulty in the mapping of the atlas content into an individual brain scan. These efforts have been headed at least in five directions: (1) direct digitization of the existing print atlases (Kall et al. 1985) ; (2) creation of bi-media atlases with both print and digital content (Zhang et al. 2003; Mai et al. 2004; Morel 2007) ; (3) 3D extension of the existing print atlases (Yoshida 1987; St-Jean et al. 1998 ); (4) creation of improved atlases derived from the print content by postprocessing, enhancements, and extensions (Nowinski et al. 1997a; Sudhyadhom et al. 2012) ; and (5) development of new electronic atlases (such as early ones, e.g., by Bohm et al. (1983) and Greitz et al. (1991) constructed from digitized crysection photographs, and many more created recently, as reviewed below). Note that the first two directions require no change in the original atlas content. To our best knowledge the first computer program with digitized (and scalable) stereotactic atlases was developed by Bertrand et al. (1974) . A digital version of the Schaltenbrand and Wahren atlas resident in a computer was created by Kall et al. (1985) . Several groups developed electronic versions of the Schaltenbrand and Bailey atlas, namely, Yoshida (1987) built a 3D atlas by interpolating print plates, a 3D volumetric model of subcortical structures was produced by Kazarnovskaya et al. (1991), and Sudhyadhom et al. (2012) created a deformable 3D atlas for deep brain stimulation surgery by employing smoothing to reduce artifacts inherent in the print version. Similarly, digital versions of the Schaltenbrand and Wahren atlas were built and incorporated into atlas-aided software systems for stereotactic and functional neurosurgery by Sramka et al. (1997) and St-Jean et al. (1998) , who developed a deformable volumetric version of the atlas. We created a multi-brain atlas database with about 1000 structures and 400 sulcal patterns embedded into a neuroimaging system (Nowinski et al. 1997a ) based on the content of four complementary classic Thieme brain atlases: Schaltenbrand and Wahren, Talairach and Tournoux (1988) , Talairach and Tournoux (1993), and Ono et al. (1990) 1 . The original atlases were highly processed, manually edited, enhanced, fully segmented and labeled, extended including into 3D, and mutually spatially co-registered. Various content representations were created, including color-coded, contour (closed for structures and open for sulcal patterns), and polygonal. They facilitate atlas use and navigation and, particularly, the unique (color-coded or contour) representation enables automated labeling. A high-quality content along with a proposed method of atlas use (Nowinski 1998 ) caused the integration of this multi-brain atlas database into a majority of surgical workstations for clinical use (Nowinski 2009) . In addition, new dedicated neurosurgical atlases have been developed in the second decade of this century and they are discussed below. Neuroeducation has driven the extension of the brain atlas content from 2D to 3D. The three-dimensional effect has been achieved by various techniques ranging from a simple form of virtual reality (VR) through QuickTime VR technology (Kling-Petersen and Rydmark 1997) to visualization of truly 3D representations by employing volume rendering of volumetric data (Hoehne et al. 1992 ) and surface rendering of geometric (polygonal) models (Nowinski et al. 2011b ). The latter approach provides fast rendering of geometric models created with subpixel resolution, e.g., as small as 1/10th of the pixel size (Nowinski et al. 2012a ). An overview of methods for 3D visualization of neuroanatomical image data and reconstruction of neuronal structures in brain atlases is presented by Maye et al. (2006) . Atlas-assisted neuroeducation, training, and simulation have greatly benefitted from the Visible Human Project (VHP) comprising the most complete volumetric data of human anatomy, including cryosection photographs, computed tomography and magnetic resonance images of American male and female specimens (Spitzer et al. 1996) . The VHP provides excellent source material for the creation of brain atlases and maps, for instance by Drury and Van Essen (1997) and Juanes et al. (2012) . The VHP additionally sparked other projects, including Chinese VHP and Korean VHP, resulting in the construction of new atlases (Zhang et al. 2003; Li et al. 2014) along with suitable tools for sectional and surface anatomy navigation as well as virtual dissection and endoscopy simulation (Chung and Park 2007) . Tremendous advancements in imaging, brain mapping, and computing propelled the development of new human electronic brain atlases. Various criteria can be employed to identify and systemize multiple directions in the content evolution of new atlases, including parcellation, modality, plurality, quality, ab/normality, lifespan, extendibility, ethnicity, spatial and temporal scales, integration, transformation, techniques of creation, and combination of them. We determine 23 directions and categorize them into eight (seven main and one combined) groups of brain atlas content development. Then, by taking into account this categorization, a brain atlas instant can be considered as an element in a seven-dimensional brain atlas space. These groups along with their component directions are as follows: 1. Scope (content extent) a. From cerebral parts (e.g., the basal ganglia (Yelnik et al. 2007) , thalamus and basal ganglia (Morel 2007) , thalamus (Krauth et al. 2010) , and deep brain structures (Lemaire et al. 2019 )) to the whole brain (Kikinis et al. 1996; Hoehne 2001; Tzourio-Mazoyer et al, 2002; Nowinski et al. 2011b ); b. From structural neuroanatomy (Rohlfing et al. 2010; Mandal et al. 2012; Nowinski and Chua 2014) to vascular neuroanatomy (Nowinski et al. 2009b; 2011a; Huck et al. 2019 ) to connectional neuroanatomy (Mori et al. 2005; Nowinski et al. 2012b; Van Essen 2013; Van Essen et al. 2013; Baker et al. 2018; Briggs et al. 2018) to gene expression (Sunkin et al. 2013) including gene expression in brain development (Kanton et al. 2019 ); c. From brain to head (Tiede et al. 1996; Chen et al. 2018) , and to head and neck (Nowinski 2017b); d. From structure to function, including functional atlases (Minoshima et al. 1994; Zhao et al. 2017; Haegelen et al. 2018; Varoquaux et al. 2018; Lehman et al. 2020) , integrated anatomic-functional atlases (Nowinski 2004; Nowinski et al. 2010) , and functional connectivity atlases (Craddock et al. 2012; James et al. 2016 ).
Parcellation Use of diverse, often multiple parcellation criteria, from classic cytoarchitecture, myeloarchitecture and gross anatomy to fMRI, chemoarchitecture (Yelnik et al. 2007 ), vascular territories (Nowinski et al. 2006) , anatomic connectivity (Mori et al. 2005) , functional connectivity (Arsiwalla et al. 2015) , anatomic-functional connectivity (Fan et al. 2016 ), (multi)receptor architecture (Amunts et al. 2010) , and/or multiplicity of them (Van Essen 2013; Glasser et al. 2016 ), among others. 3. Modality a. From postmortem to in vivo data (Lehmann et al. 1991; Nowinski et al. 2015a; Dickie et al. 2017; Oishi et al. 2019 ); b. Integrating postmortemin vivo data (Nowinski et al. 1997b; 2002b; Yelnik et al. 2007; Cho et al. 2008; Amunts et al. 2014 ); c. Increased teslage, from 1.5T (Tesla) (Hoehne 2001) to 3T (Nowinski et al. 2009b; Rohlfing et al. 2010 ) to 7T (Cho et al. 2008; Nowinski et al. 2015a; Saygin et al. 2017; Huck et al. 2019; Liu et al. 2020 ) to 9.4T (Yushkevich et al. 2009 ); d. From image to non-image data, transforming into brain atlases non-image data, such as stimulating electrode geometry (Nowinski et al. 2003) and neurologic parameters (Nowinski et al. 2014a) . 4. Plurality a. Specimen-related: from a single specimen to population atlases for cerebral parts (such as the cerebellar nuclei (Dimitrova et al. 2006) , insula (Faillenot et al. 2017) , cortical structures (Shattuck et al. 2008) , and cerebral arteries (Dunås et al. 2017) to the whole human brain (Mazziotta et al. 1995; 2001; Thompson et al. 2000) ; b. Variant-related: from a single variant to a collection of variants, for instance, the cerebrovascular variants (Nowinski et al. 2009a ); c. Modality-related: from uni-modal to multi-modal atlases with the use of multi-modal complementary data (e.g., Johnson and Becker 1999; Toga et al. 2006; Nowinski et al. 2010; Hawrylycz et al. 2012; Ding et al. 2016 ); d. Channel-related: e.g., with anatomy, diffusion, and tissue channels (Rohlfing et al. 2010 ); e. Atlas-related: from a single atlas to arrays of fully parcellated atlases or mega multi-atlases (Wu et al. 2016) . 5. Scale a. Spatial scale, from macro-to meso-to micro-to nanoscales along with integrating atlas data across multiple spatial scales (Assaf et al. 2013; Ding et al. 2016; Ecker et al. 2017 ); b. Temporal scale covering atlases from development (Kanton et al. 2019) to lifespan including age-matched atlases to accommodate age-dependent anatomical changes ranging from pediatric to geriatric populations (Wu et al. 2016; Zuo et al. 2017; Zhang et al. 2018; Oishi et al. 2019) ; c. Integrating spatio-temporal scales (Sunkin et al. 2013; Bozek et al. 2018 ).
Ethnicity Ethnic-specific atlases, for instance, for Chinese (Zhang et al. 2003) , Korean (Cho et al. 2008) , and Caucasian (Nowinski 2017b) specimens.
Abnormality From normal to disease-specific atlases for various brain disorders, for instance, Alzheimer's disease (Thompson et al. 2001) , dementia (Mega et al. 2005) , and stroke (Nowinski et al. 2014a (Lalys et al. 2010) . We witness recently an enormous development of population-based brain atlases both in health and disease. Population-based structural atlases have been built for the whole brain (Liang et al. 2015; Wu et al. 2016 ) and its specific regions, such as the cortical areas (Shattuck et al. 2008; Glasser et al. 2016) , cerebellum (Diedrichsen et al. 2009) , brainstem (Meola et al. 2016) , subcortical nuclei (Pauli et al. 2018) , thalamic nuclei (Iglesias et al. 2018; Najdenovska et al. 2018) , insula (Faillenot et al. 2017) , some gyri including the parietal lobe gyri (Wild et al. 2017 ) and the inferior frontal gyrus (Hammers et al. 2007) , and venous cerebrovasculature (Huck et al. 2019) . More advanced atlases have been developed in terms of population (Liang et al. 2015) , specimen age range span (Wu et al. 2016; Zhang et al. 2018) , and age appropriateness (Fonov et al. 2011) . For instance, the atlas of Chinese adults contains a large number of 2020 specimens whose age spans from 20 to 75 years at a 5-year interval (Liang et al. 2015) . The longitudinal atlas for normative brain development and aging spans the age range of 1-83 years, while the quantitative susceptibility mapping used for its creation may facilitate the estimation of age-related iron changes in deep gray matter nuclei and myelin changes in white matter (Zhang et al. 2018) . A mega multi-atlas (Wu et al. 2016 ) constitutes an inventory of 90 brain atlases with the specimens ranging from 4 to 82 years of age. Several agedependent brain atlases have been built also for children (Ou et al. 2017; Bozek et al. 2018) and fetuses, such as a dynamic 4D (four-dimensional) probabilistic atlas of the developing brain (Kuklisova-Murgasova, et al. 2011) . The baby brain atlases developed for specimens younger than 12 months old (for the fetus, neonate, and infant) are reviewed by Oishi et al. (2019) . Besides probabilistic structural atlases also a variety of probabilistic connectional atlases (Meola et al. 2016; Figley et al. 2017; Yeh et al. 2018; Chenot et al. 2019) , functional maps and atlases (Nowinski et al. 2003; Nowinski 2009; Breshears et al. 2015) , and vascular atlases (Dunås et al. 2017; Bernier et al. 2018; Mouches and Forkert 2019) have been created. Developments in mapping the microscopical organization of the brain along with the progress in nanoscience (Alivisatos et al. 2013) enable the construction of brain maps and atlases across spatial scales extending from macro to meso to micro to nano. Examples include the BigBrain with 20-micrometer resolution (Amunts et al. 2013 ), a comprehensive cellular-resolution (of 1 μm/pixel) brain atlas linking macroscopic anatomical and microscopic cytoarchitectural parcellations (Ding et al. 2016) , the Brain Activity Map as the functional connectome to elucidate emergent levels of neural circuit function (Alivisatos et al. 2012) , a temporal cell atlas of gene expression in brain development (Kanton et al. 2019 ), a genomics brain atlas (Sunkin et al. 2013) , a proteomic brain atlas (McKetney et al. 2019) , an atlas of serotonin (Beliveau et al. 2017) , and an atlas of brain transcriptome (Hawrylycz et al. 2012) . In particular, identifying the different brain cell types to determine their roles in health and disease is of great importance and it is established as one of the six goals of the BRAIN Initiative (BRAIN Working Group 2014). Toward achieving this goal a whole-brain cell atlas is under development by Ecker et al. (2017) that integrates molecular, anatomical, and physiological annotations of neuronal cell types for a comprehensive characterization of cell types, their distributions, and patterns of connectivity.
Evolution of Brain Atlas Applications The rationale of creating the early cortical maps, the result of human curiosity, was to represent the knowledge of new discoveries about the human brain. The brain knowledge capturing, aggregation, and representation by means of human brain atlases has been the first application of the atlases, and this central role remains until the present. Research has been the dominant application of human brain atlases (Roland and Zilles 1994) employed as tools for analysis of brain structure and function (Hess et al. 2018) , means to integrate neuroscience research data from healthy and diseased brains to increase data sharing and reusing (Bjerke et al. 2018) , and a potential tool suitable for image structurization through atlas-based image parcellation to utilize a vast amount of imaging information available in medical record systems, such as the PACS (picture archiving and communication system) (Mori et al. 2013) . Moreover, disease-specific atlases, such as (Thompson et al. 2001; Mega et al. 2005; de Haan and Karnath 2017) facilitate quantification of brain structural deficits in epilepsy, depression, schizophrenia, Alzheimer's disease, bipolar disorders, autism and others disorders as discussed by Toga and Thompson (2005) . Human brain atlases are also useful beyond research in medical education and clinical applications. Stereotactic and functional neurosurgery was the first, major clinical application of brain atlases. We observe that every two decades mark major progress in this field. The first print atlases were created in the 1950th, the first digitized brain atlas was developed in the 1970th (Bertrand et al. 1974) , and the acceptance of our electronic brain atlases to clinical practice by the community (and 13 surgical companies) started in the 1990th (Nowinski 2009) . Initially, a digital atlas was used off-line for referencing while being placed beside the displayed patient-specific scan. In this way, for instance, The Electronic Clinical Brain Atlas (Nowinski et al. 1997b ) had been employed next to a surgical workstation to plan neurosurgery before our brain atlas database was directly integrated with surgical workstations, such as the StealthStation (Nowinski 2009 ). In the second decade of this century several novel, neurosurgery-dedicated atlases have been developed for electrode placement in deep brain stimulation (Sadikot et al. 2011; Dergachyova et al. 2018; Haegelen et al. 2018; Nowacki et al. 2018 ). In the pre-tomographic era, stereotactic brain atlases were useful to localize deep stereotactic targets. The introduction of diagnostic imaging has not eliminated brain atlases but rather changed their role and function (Nowinski 2009) . Namely firstly, a high atlas parcellation, typically greater than that of a scan, allows the individualized atlas to facilitate targeting. Secondly, extensive atlas features in combination with its ease of use and precision facilitate neurosurgery planning and provide intraoperative support, like those available in The Cerefy Clinical Brain Atlas: Extended Edition with Surgery Planning and Intraoperative Support (Nowinski et al. 2005a ). Thirdly, several new dedicated brain atlases have been created that are derived from various modalities including histology (Chakravarty et al. 2006) , electrophysiology (Finnis et al. 2003; Nowinski et al. 2003) , and multi-modalities (Yelnik et al. 2007; Nowinski et al. 2010; Haegelen et al. 2018) . Other examples of atlas use in neurosurgery include a digital brain atlas for surgical planning (Kikinis et al. 1996) , an Internet portal for stereotactic and functional neurosurgery shifting the paradigm in atlas building from manufacturercentric (dependent) to neurosurgical community-centric (Nowinski et al. 2002a) , and a practical 3D atlas for a preoperative white matter-specific planning of subcortical trajectories (Jennings et al. 2018) . Several neuroeducational atlases were created in the 1990th, the Decade of the Brain, including BrainStorm (Dev et al. 1992 ), Digital Anatomist (Sundsten et al. 1994) , A.D.A.M. (A.D.A.M 1996), Microvascular Atlas of the Head and Neck (Bayer 1996) , The BRAIN project (Kling-Petersen and Rydmark 1997), and The Electronic Clinical Brain Atlas (Nowinski et al. 1997b ). These initial efforts were followed by the development of more advanced atlases in terms of content and functionality, such as Voxel-man (Hoehne 2001) , The Cerefy Atlas of Brain Anatomy (Nowinski et al. 2002b ), Primal's Interactive Head & Neck (Berkovitz et al. 2003) , The Cerefy Clinical Brain Atlas (Nowinski and Thirunavuukarasuu 2004) , The Cerefy Atlas of Cerebral Vasculature (Nowinski et al. 2009b) , The Human Brain in 1492 Pieces (Nowinski et al. 2011b) , The Human Brain in 1969 Pieces: Structure, Vasculature, Tracts, Cranial Nerves, Systems, Head Muscles, and Glands (Nowinski and Chua 2014), The Human Brain, Head and Neck in 2953 Pieces (Nowinski et al. 2015a) , and the Human Anatomy Atlas (Visible Body n.d.). In addition, individualized atlases that parcellate and annotate brain scans are useful for the creation of teaching files of brain anatomy and function (Oishi et al. 2019) . The brain atlases also play a role in training and simulation, e.g., in neurosurgery (Serra et al. 1997 ) and radiotherapy (Roniotis et al. 2012) . Human brain mapping in research and clinical practice is another major area of brain atlas employment. Digital brain atlases are exploited here to provide the underlying neuroanatomy and to automatically label activation loci in functional images with cortical areas and stereotactic coordinates. Application examples include the BrainMap (Lancaster et al. 2000) and the Brain Atlas for Functional Imaging (Nowinski et al. 2000b ). Both these tools employ digital Brodmann's areas (derived from the Talairach and Tournoux (1988) brain atlas) that are hidden in the BrainMap while explicitly available and displayed to the user in the Brain Atlas for Functional Imaging. Brodmann's areas, despite being one century old and originally limited to two views on the visible part of the cortical surface only, are still today applicable references in human brain mapping to register functional activations to the underlying anatomy (Amunts and Zilles 2015). ). Because of well-known limitations of the Talairach and Tournoux atlas (see, e.g., (Nowinski and Thirunavuukarasuu, 2009) ) in order to improve labeling of functional foci a dedicated AAL (Automated Anatomical Labeling) atlas was developed from a T1-weighted scan with 45 anatomical volumes of interest in each hemisphere (Tzourio-Mazoyer et al, 2002) . Nuclear medicine, such as SPECT (single-photon emission computed tomography) and PET (positron emission tomography), produces images of relatively poor spatial resolution, which makes it difficult to relate the functional information contained there to the corresponding underlying neuroanatomy. In order to enhance the accuracy and consistency of the anatomic interpretation of PET functional brain images, Minoshima et al. (1994) constructed a PET stereotactic brain atlas from a high-resolution [18F]FDG (fluorodeoxyglucose) images of a normal volunteer. In addition, to assist in the interpretation of SPECT scans of the brain, a 3D neuroanatomical atlas was created from an MRI scan of a normal, healthy volunteer by Lehmann et al. (1991) . Human brain atlases have potential in stroke management for prediction, diagnosis, and tr eatment (Nowinski 2020) . The atlases of anatomy and blood supply territories support decision making in thrombolysis and provide a quantitative assessment of the infarct and penumbra (Nowinski et al. 2006 ). These two atlases also facilitate rapid and automatic detection, localization, and classification of ischemic and hemorrhagic lesions in the emergency room (Nowinski 2020) . The probabilistic stroke atlas, created by the integration of brain scans with textual neurologic parameters of previously managed stroke patients, enables prediction of stroke outcomes (Nowinski et al. 2014a) . We have also developed atlas-based applications in several other areas, including neuroradiology, neurology, psychology, psychiatry, and proposed new solutions in some niche applications, such as atlas-guided do-it-yourself neurosurgery suitable for patients (Nowinski 2009 ) and an atlas-enhanced operating room for the future (Benabid and Nowinski 2003) . In neurology, the 3D Atlas of Neurologic Disorders (Nowinski et al. 2014b) facilitates the understanding of neurologic deficits resulting from brain damage. The atlas bridges neuroanatomy, neuroradiology, and neurology (Nowinski and Chua 2013a) . It serves as an educational means for neurology students and residents as well as a reference for neurologists. This atlas is also a potentially useful tool for psychologists, and particularly neuropsychologists, to communicate with patients. The Cerefy Neuroradiology Atlas (Nowinski and Belov 2003) available over the Internet contains a fully segmented and labeled anatomic brain atlas. It provides functions for a rapid atlas-to-scan registration, interactive structure labeling and annotating, and mensuration. To our best knowledge, this is the first online, publicly available atlas-based application for neuroradiology. In general, brain atlases have a still unexploited potential in neuroradiology. For instance, in (Nowinski 2016) nine various scenarios of atlas use in neuroradiology were discussed based on the earlier developed working prototypes ranging from image interpretation to reporting to dealing with data explosion and to communication (for both doctor-todoctor and doctor-to-patient). In psychiatry, we employed a brain atlas to automatically generate anatomic volumes of interest for subsequent analysis in a population of schizophrenic patients and controls to study the passivity phenomenon (Sim et al. 2009) .
Development of Atlas Functionality The early bare brain maps and atlases had no or a very limited supporting functionality. Therefore, certain early print stereotactic brain atlases, besides providing standard anatomical indices (along with some accompanying textual description), were placed in a stereotactic coordinate system enabling localization and referencing. Moreover, certain atlases were equipped with transparent overlays with structure annotations over the brain plates to facilitate structure delineation and identification. Conceptually, this simple functionality already signaled the necessity of equipping brain atlases with suitable tools enabling their clinical applications. This necessity in the pre-digital era was clearly expressed by a popular practice performed in the operating room of generating resized (individualized) brain atlas plates by means of an overhead projector and drawing a planned stereotactic trajectory directly on the displayed projection. The need of generating individualized atlases in neurosurgery was met in the first computer program with digitized stereotactic atlases developed by Bertrand et al. (1974) that provided 1D (one-dimensional) atlas scaling along the intercommissural distance. This solution was followed by a 3D piece-wise linear (Nowinski et al. 2000a ) and non-linear (Ganser et al. 2004 ) brain atlas warping. The requirements from stereotactic and functional neurosurgery have been an initial major driving force behind the brain atlas development, both in terms of content (as discussed above) and functionality. Specific atlas-related tools have been proposed for preoperative planning, intra-operative support, and postoperative assessment (Nowinski 2001a) . Pre-operatively, the atlas facilitates the target and trajectory planning to avoid some critical structures (such as the optic tract), and provides the list of trajectory-intersected structures. In order to increase both the quality of planning and the surgeon's confidence, multiple complementary atlases are employed (Nowinski et al. 2000a; 2010) . In general, the atlas facilitates the planning of the access corridor to any target structure by determining all the structures encountered along the selected corridor and those neighboring it, allowing the neurosurgeon to assess various potential corridors in the process of decision making. Intra-operatively, the atlas provides the actual structure where the tip of the electrode is located, the list of structures already intersected by the electrode, distances to critical structures, and the surrounding anatomic and vascular context (Nowinski et al. 2010) . Additionally, the probabilistic functional atlas makes the targeting more accurate by determining the best location within the whole target structure (Nowinski et al. 2003; 2005b) . Post-operatively, the atlas facilitates to analyze the correctness of placement of a stimulating electrode or a permanent lesion. The first (to our best knowledge) collaborative use and construction of a brain atlas over the Internet by the neurosurgical community was offered in the portal for stereotactic and functional neurosurgery supporting a probabilistic functional atlas (Nowinski et al. 2002a ). This atlas is calculated from neuroelectrophysiologic and neuroimaging patient-specific data acquired during functional neurosurgical procedures (Nowinski et al. 2003) . The portal supports the atlas with the functionality enabling data uploading to the central database or downloading locally in order to combine them with the neurosurgeon's own data, followed by the calculation of the individualized probabilistic functional atlas and surgery planning. The atlas is displayed graphically in 2D, 3D, and as a probability distribution histogram along with the data tree (including patients, electrodes, contacts, and coordinates) in a text format. The development of atlas functionality has also been driven by the needs of the human brain mapping community, mainly for the integration of structural and functional, and, generally, multi-modal images in the same stereotactic space as well as for atlas-assisted automatic labeling of activation loci in functional images with cortical areas and stereotactic coordinates. The SPM anatomy toolbox (Eickhoff et al. 2005 ) is an example of an image integration tool that enables the combination of probabilistic cytoarchitectonic maps and results of functional imaging studies. Another data integration tool is the Neuroinformatics Platform within the Human Brain Project (Bjerke et al. 2018) . Examples of labeling tools are the BrainMap (Lancaster et al. 2000) and the Brain Atlas for Functional Imaging (Nowinski et al. 2000b ). The BrainMap assigns a label of the cortical area closest to the examined activation locus. This process is blind to the user as the brain atlas employed is hidden from display. In the Brain Atlas for Functional Imaging the parcellated, labeled, and color-coded cortical areas are explicitly available and displayed to the user, who has full control over the process of activation loci labeling and is able to edit their positions if needed. This atlas processes functional images through a locus-driven analysis (Nowinski and Thirunavuukarasuu 2003) . The activation loci in functional images are extracted automatically by thresholding with the option of interactive editing. Then, the atlas of anatomy extended with Brodmann's areas is employed for labeling of the activation loci with the names of cerebral structures, Brodmann's areas, and stereotactic coordinates. The activation loci are marked on the images with the superimposed atlas, and the list of all labeled loci along with their values on the anatomic and functional images is provided to the user. Neuroinformatics tools and repositories also have been developed to store various and heterogeneous results of analyses. For instance, NeuroVault.org stores these results in a form of statistical maps, parcellations, and atlases (Gorgolewski et al. 2016) ; and BALSA is a database of the brain analysis library of spatial maps and atlases (Van Essen et al. 2017) . Construction of population atlases as well as atlas-assisted neuroimage processing and analysis in any application requires atlas-to-scan (or scan-to-atlas) registration. Pioneering work on atlas-to-scan elastic registration was done by Bajcsy et al. (1983) and Gee et al. (1993) . Brain image registration algorithms are evaluated, for instance, by Klein et al. (2009) and Ou et al. (2014) . Image registration is also employed for multi-modal atlas construction and atlas-guided segmentation of brain images. Automatic segmentation of brain images, in particular, is of great importance and it can be performed through atlas-to-scan registration, as the individualized atlas segments and labels the underlying neuroanatomy. The majority of methods are for the segmentation of structural neuroanatomy, though some approaches are developed to provide atlas-based processing of connectional neuroanatomy (Labra et al. 2017 ) and cerebrovascular anatomy (Passat et al. 2006; Dunås et al. 2016) . As multi-atlases are more powerful than single atlases, numerous approaches have been developed for multi-atlas based brain segmentation (Aljabar et al. 2009; Artaechevarria et al. 2009; Lötjönen et al. 2010; Wu et al. 2016; Zaffino et al. 2018; Li et al. 2019) . In neuroeducation a typical atlas-related functionality includes labeling, searching, and atlas display and manipulation. Beyond typical operations, some atlases provide more sophisticated operations, such as advanced labeling with vessel diameters (Nowinski et al. 2011b ) and pathology description (Nowinski et al. 2014b ) as well as quantification, such as geometric measurements (Nowinski et al. 2015a) . The atlas also enables automatic testing suitable for both self-testing and classroom testing. A testing module for atlasenabled evaluation of brain knowledge in neuroeducation was designed and incorporated into The Cerefy Atlas of Brain Anatomy (Nowinski et al. 2002b) , and the corresponding method presented in (Nowinski et al. 2009c ). The module allows the instructor to set the testing parameters first, such as the scope of the tested knowledge, scoring points, and the number of attempts. The items (structures) in the index are consecutively numbered forming a list. A random generator selects randomly items from the list while avoiding repetition. There are two types of queries "Where is?" and "What is?" to test location and naming of cerebral structures, respectively. When the name of the selected item is highlighted in the index, the student is tested against "Where is?" aiming to point to the selected structure in the atlas (image or model). When the selected structure is highlighted in the atlas, the student is tested against "What is?" aiming to indicate the name of this structure in the index. After all the structures have randomly been selected, the module provides the total score and the time spent to perform the test. Note that for the same scope of a tested brain knowledge the queries, which are randomly generated, are different each time avoiding this way the situation that the student copies someone else's answers or memorizes them. Some other examples of application-specific functionality in our atlas-based solutions include brain scan interpretation in neuroradiology (Nowinski and Belov 2003) , segmentation and labeling of pathological neuroimages (Nowinski and Belov 2005) , automatic generation of atlas-derived regions and volumes of interest (VOI/ROI) for fast comparison of the left and right cerebral hemispheres to detect pathology (Nowinski 2020 ) and for statistical analysis in populations (Sim et al. 2009) , aggregation of image and clinical brain data (Nowinski et al. 2014a) , dealing with data explosion (Nowinski 2016) , radiology reporting (Nowinski 2016) , and brain knowledge communication (for both doctor-to-doctor and doctor-to-patient) (Nowinski 2016) . The abovementioned operations and tools are incorporated into the atlasing software platforms, mostly to enable and enhance the atlas use. However, there exist numerous stand-alone tools suitable for atlas creation and use that are not incorporated directly into the created brain atlas platforms, such as FreeSurfer, a suite of tools for a cortical surface generation and quantification of functional, connectional and structural properties of the human brain (Fischl 2012) extended recently with the probabilistic atlas of the thalamic nuclei (Iglesias et al. 2018) ; SPM for a neuroanatomical variability assessment (Ashburner 2009) ; FSL, a comprehensive library of analysis tools for functional, structural, and diffusion MRI brain imaging data (Jenkinson et al. 2012) ; the Medical Imaging Interaction Toolkit (MITK) integrating two other powerful toolkits, the Visualization Toolkit (VTK) and the Insight Toolkit (ITK) (Wolf et al. 2005) ; and the Vascular Editor to create and edit vascular and, generally, tubular-like such as cranial nerve networks (Marchenko et al. 2010) . Our experience shows that the tools directly integrated with the atlas have proved their value allowing any new atlas modules to be created and edited within the already existing neural context (Nowinski et al. 2012a; b) . Recently, a new generation of methods, tools, repositories, and neurotechnologies is planned or already under development. For instance, intensive technology development and validation is outlined under the BRAIN Initiative (BRAIN Working Group 2014; Jorgenson et al. 2015) . Numerous atlas-related tools are being developed within brain big projects. For instance, a common automated preprocessing framework has been developed within the Human Connectome Project to bring multiple magnetic resonance imaging modalities together across a large coh o r t o f s u b j e c t s ( G l a s s e r e t a l . 2 0 1 3 ) . T h e Neuroinformatics Platform within the Human Brain Project develops tools to facilitate data acquisition and annotation, assignment of the anatomical location to data, and assembly of and access to spatially indexed information (Bjerke et al. 2018) . Other examples of such tools developed within the Allen Brain Atlas, BigBrain, and FSL atlas, among others, are given in (Amunts and Zilles 2015) . The Scalable Brain Atlas is a collection of web services that provides unified access to a large collection of public brain atlasing resources for the human and non-human species (Bakker et al. 2015) . Therefore, despite relatively slow progress in the development of the brain atlas enabling functionality so far in comparison to that of the atlas content, the recent brain big projects will strongly drive this functionality development due to dramatic needs to handle big data including their storage, visualization, processing, analysis, and (most importantly) interpretation. Moreover, technology advancement will enhance the brain atlas functionality development to create new atlases, such as a holographic brain atlas (Petersen et al. 2019 ) (note that much earlier we proposed to use holography in an atlasenhanced operating room for the future (Benabid and Nowinski 2003) ).
Evolution of Atlas Availability The human brain atlases reviewed above are available to the community in various ways and this availability can be considered in terms of what is available and how it can be accessed. The atlases are available on two major media, print and electronic, resulting in three categories: print atlases, electronic atlases on various platforms, and transitional atlases from the print to the electronic medium. The early maps and atlases were available in a print form. The transition from the print to electronic medium has been done via two channels by (1) creating both print and electronic (bi-media) atlases, and (2) derivation of early electronic atlases from print materials by direct atlas plate digitization with no content change or with content extension by postprocessing and enhancement. Tremendous developments in computing enable almost an unlimited growth of electronic brain atlases on numerous platforms ranging from mobile solutions to leading-edge supercomputers. Electronic brain atlas platforms can generally be classified along multiple divisions: stand-alone versus webbased; stationary versus mobile; low-cost versus highend workstations; with standard interaction and display versus VR-enhanced, augmented reality (AR)-enhanced and holographic display; standard computer versus supercomputer; and single computer versus computer clusters, networks, and cloud computing. In neuroscience research, it is usually required to provide a brain atlas within a web-based solution. In general, brain atlases can run on various platforms. For instance, we have developed electronic brain atlases available and running on several platforms, including stand-alone plug-in library (Nowinski 2009) ; workstation (Nowinski 2009) ; notebook and desktop (for Windows and MAC) (Nowinski et al. 2005a; 2011b; Nowinski and Chua 2014) ; Internet-based (Nowinski et al. 2002a) ; mobile (iPhone (Nowinski et al. 2009c) , iPad (Nowinski and Chua 2013b) , Android (Nowinski et al. 2014c) ; and VRenabled (Serra et al. 1997) . It is worth mentioning that the latter pioneering 3D brain atlas, employing a VR environment with a 3D natural interaction and stereoscopic display, was completed as early as in 1997. Brain big projects, however, such as The Human Brain Project, require leading-edge supercomputers (Amunts et al. 2016) . From a user's standpoint, we distinguish three levels of accessibility: non-accessible, private (with limited or unlimited access), and public (with registered or unregistered access; free or payable). The non-accessible level means that the atlas is published by its creators and available only to them, and the community is aware of the atlas but has no access to view it completely nor use it. This is probably the most common situation. Private access indicates accessibility of the atlas to a certain group of users, such as members of a consortium. Public access implies that any user may have access to the atlas after meeting certain condition(s), such as registration and/or payment. For instance, the print atlases are public, payable with unregistered access. Most educational electronic brain atlases are public and payable, such as Voxel-man (Hoehne 2001) , The Human Brain in 1492 Pieces (Nowinski et al. 2011b ), Focus Digital Anatomy Atlas. Neuroanatomy running on iPhone and iPad (Focus Medica), and Human Anatomy Atlas (Visible Body n.d.). Our latest and most advanced atlas The Human Brain, Head and Neck in 2953 Pieces (Nowinski et al. 2015a ) is public, free of charge with registration required by its publisher at http://www. thieme.com/nowinski/. Although restricted access may constrict in some cases atlas availability, we may guess that overall this availability substantially grows over time, as the numbers of both atlas creators and their users have been rising tremendously. This guess is corroborated by Table 1 that provides the numbers of publications over time cited on PubMed under the term "human brain atlas" indicating the growth over 470 times from 1 publication in the year 1950 to 474 publication in the year 2018. Between years 2010-2018 this growth was almost 12 fold. The overall number of citations is 4350. The same term "human brain atlas" searched on Google Scholar gives "about 762,000 results".
Generations of Human Brain Atlases Observing the evolution of human brain maps and atlases, four atlas generations can be distinguished, namely: (1) early cortical maps, (2) print stereotactic atlases, (3) early digital atlases, and (4) advanced brain atlas platforms. From a timeframe standpoint, approximately the first atlas generation was developed in the first half of the 20th century (with the major maps published in the first three decades), the second generation in the second half of the 20th century (with the majority of atlases published in its first four decades), the third generation in the Decade of the Brain (and a handful of atlases a little earlier), and the fourth generation in the 21st century, the century of the brain and mind. The above review, generations of atlases, and their present state are summarized in a form of the human brain atlas evolution diagram in Fig. 1 .
Discussion The human brain atlases have been evolved tremendously, especially in recent decades, in multiple directions, as captured diagrammatically in Fig. 1 . This evolution has been driven by sophisticated imaging techniques, advanced brain mapping methods, vast resources of brain data accumulated at an unprecedented rate, analytical strategies, and powerful computing. The effects of this explosive growth span from a few hand-drawn maps to multi-atlases, from print editions to web-based repositories, from 2D to nD, from determinist to probabilistic, from unimodal to multi-modal, from a cortical organization to an all-level brain organization from genes to the whole brain, from normal to pathologic, from gross to nanoscales, and various combinations of these above, among others. Several papers and reports have addressed the future trends that can be expected in the human brain atlas evolution, mainly in terms of an atlas content (Toga et al. 2006; Evans et al. 2012; Amunts et al. 2014; BRAIN Working Group 2014) . The brain atlases have been employed in a wide spectrum of applications and their usefulness depends not only on the atlas content, but also on functionality and availability. Hence this review has been conducted from these four perspectives: content, applications, functionality, and availability, in contrast to other works limited mostly to atlas content. Content-wise, the human brain atlases have evolved from a few hand-drawn maps to an atlas as a collection of maps and images; to multi-atlases; to repositories of multi-modal brain images in health and disease; to heterogeneous databases; to composable, manipulable and explorable 3D and, generally, nD cerebral models; to platforms for brain knowledge aggregation and integration; to brain atlas data at macro, meso, micro, nano, and hybrid scales with the resolution ranging from the whole brain to synapses; and to large databases with massive amounts of data aiming to discover knowledge being developed within multi-center and/or multi-national projects and initiatives. In an over century-long process of the human brain map and atlas creation, we have distinguished four generations: (1) early cortical maps (created in the first half of the 20th century), (2) print stereotactic atlases (published in the second half of the 20th century), (3) early digital atlases (produced predominantly in the Decade of the Brain), and (4) advanced brain atlas platforms (being developed in this century). We also noticed that every two decades mark major progress in the human brain atlas evolution. The first stereotactic brain atlases were created in the 1950th, the first digitized brain atlas was developed in the 1970th, the introduction of electronic brain atlases to clinical practice began in the 1990th followed by an explosion in brain atlas development propelled by the brain big projects that started in the 2010th . The development of electronic brain atlases spans the last two generations and in this area we have distinguished five avenues. The recent and most prominent avenue is the creation of new electronic brain atlases. We give numerous examples of vast activities in the area of atlas content development heading in at least 23 various (though non-exhaustive) directions, which are categorized in eight groups taking into account scope (content extent), parcellation, modality, plurality, scale, ethnicity, abnormality, and a mixture of them. Application-wise, brain atlases are employed in education, research, and clinical practice. The role and usefulness of the brain atlases have been expanding both within the research area and beyond it. The main atlas application area is research and brain knowledge gathering ranging from knowledge capturing to knowledge aggregation to knowledge discovery. Other areas of atlas applications include human brain mapping (spanning research and clinical practice), stereotactic and functional neurosurgery, neuroeducation, and specific areas, such as neuroradiology, neurology, psychology, stroke, and psychiatry. The major application-wise shift has been from research to clinical practice, particularly in stereotactic and functional neurosurgery to treat patients. A brain atlas importance and Fig. 1 A human brain atlas evolution diagram with four generations and four categories: content (only for the new electronic atlases), applications, functionality, and availability, each subsequently divided into sub-categories potential in clinical applications have been raised and addressed by a few authors (Mori et al. 2013; BRAIN Working Group 2014) . In fact, the development of brain atlas-based clinical applications for prediction, diagnosis, and treatment has been a major focus of our work. Neurosurgery planning and assessment (Nowinski 1998; 2001; 2009; Nowinski et al. 2010 ) was our first clinical application with anatomic, functional, and vascular atlases created. Our solutions in stereotactic and functional neurosurgery have been licensed to 13 surgical companies and integrated with surgical workstations of the leading companies, namely, Medtronic, Brainlab, and Elekta (Nowinski 2009 ). In addition, we have developed working prototypes in other fields for atlas-assisted brain pathology detection (Nowinski 2020) , quantification of cerebral lesions (Nowinski et al. 2006) , segmentation and labeling of pathological neuroimages with tumors causing a mass effect in brain cancer (Nowinski and Belov 2005) , stroke management (Nowinski et al. 2006; Nowinski 2020) , and stroke outcome prediction (Nowinski et al. 2014a) . A vast, still unexploited, potential of brain atlas in neuroradiology has been addressed in (Nowinski 2016) describing nine applications for which working prototypes (proofs of concept) we developed earlier and presented at clinical meetings. However, despite several examples of brain atlas use in clinical applications (as products or working prototypes), these applications are still lagging behind the progress in the development of the atlas content. One of the main obstacles in introducing the brain atlas solutions to clinical practice is their validation, which is tedious, time-consuming, and costly; particularly, clinical validation is beyond a reach of a research lab because of its high cost. In contrast to the content-wise atlas development being widely carried out by numerous groups as well as by national and multi-national consortia, the development of atlas functionality has been relatively neglected, until recently as the problem of managing data explosion requires powerful, suitable, and dedicated tools. The early atlases evolved from bare, hand-drawn maps to print stereotactic atlases with images scalable using an overhead projector to electronic deformable atlas platforms to VRand AR-enhanced atlases to atlas-engines meaning the atlases serving as tools by themselves that support brain data management, neuroimage processing and analysis, decision making, and knowledge discovery. From an application standpoint, the tools providing an atlas with its supporting functionality belong to three categories: (1) educational tools to explore the atlas, test knowledge, and prepare teaching materials (that can be grouped as student-oriented, educator-oriented, self-testing, and a mixture of them); (2) research tools enabling brain investigation and knowledge discovery; and (3) clinical tools to allow the clinicians to better prevent, diagnose, treat, and cure brain diseases. Efficient and user-friendly tools are, in particular, required, in education. We have attempted to develop new education tools going beyond those available in standard educational atlases, such as Voxel-man (Hoehne 2001) or Interactive Head & Neck (Berkovitz et al. 2003) . These tools enable novel educational use of the atlas, such as self-testing and classroom assessment (Nowinski et al. 2009c ) available on notebooks and mobile devices, interdisciplinary education across neuroanatomy-neuroradiology-neurology (Nowinski et Chua 2013a) , advanced education for residents and clinicians with a user's "de/composable" content and context (Nowinski et al. 2014b; 2015a) , and patients' education and instruction (Nowinski 2016) . From a usage standpoint, atlas tools can be classified into two broad categories: general and specific. General tools support typical atlas-enabled operations, such as segmentation, labeling, manipulation, quantification, and querying. Specific operations are those customized to a certain field and/or particular use, such as automatic testing, generation of teaching materials, ROIs/VOIs generation and analysis, targeting, safety analysis, postoperative assessment, locusdriven analysis, decision making support, prediction of occurrence and outcomes, scan interpretation, knowledge communication, and a combination of them. From an integration standpoint, atlas-related tools can be stand-alone or directly integrated with the atlas platform. Availability-wise, the major developmental step was obviously from print to digital atlases. Enormous progress in computing enables almost unlimited development of digital brain atlases to run on numerous platforms ranging from mobile solutions to notebooks to interactive web-based visualization platforms to VR/AR systems to high-end workstations to c om pu t e r cl us t e r s , ne t w o r k s , a n d le ad i n g -e dg e supercomputers. The atlas availability substantially grows over time with the numbers of both atlas creators and their users tremendously raising. If approximated by the growth of the human brain atlas publications on PubMed, the atlas availability growth from the year 1950 to the year 2018 would be over 470 times. This work has several limitations. We have tried our best to make this state-of-the-art review in the human brain atlas evolution as complete as possible. However, the overall number of publications about this subject on PubMed is vast of 4350 (and about 762,000 references on Google Scholar) and rapidly growing, which makes a fairly complete state-of-the-art review quite difficult (if possible at all). In some areas, such as clinical applications, any relevant research publications may simply not exist, and the names of atlas creators and developers may not be disclosed by the providers to the atlas users (as, for instance, is in the case of our brain atlases licensed to surgical companies). The brain atlas content evolution is divided at two levels into 8 groups at the first and 23 directions at the second level. We believe that the categorization of the atlas content development into these 8 groups covers the whole landscape, though it is not unique and other criteria might be applied. This categorization is neither distinctive and some groups may overlap, for instance, the increasing scale may result in the increasing scope. The overall 23 directions in the atlas content development are not exhaustive and could be finer, especially in the last (combined) group. Likewise, the ethnicity and abnormality groups could be subdivided into directions, each for a specific ethnicity or disease, respectively. The approximation of the atlas availability growth through the number of publications about human brain atlas may be underestimated even by a few orders. Usually a publication about a free atlas attracts a plethora of its users, and even the number of citations may not be representative. For instance, our free brain atlas (Nowinski 2017b ) has a download-tocitation ratio of 160. This review is restricted to the human brain atlases. Several authors have overviewed non-human brain atlases for various species, including primates (marmoset, mouse lemur, squirrel monkey, macaque, and chimpanzee by Thiebaut de Schotten et al. ( 2018 )), rodents and marsupials (rat, mouse, and opossum by Bakker et al. (2015) ), and other animals by Hess et al. (2018) . It is also worth mentioning that several human spinal cord atlases have been constructed, for instance, by Taso et al. (2013) and Lévy et al. (2015) . Finally, this review reflects a personal perspective and three-decade-long experience of the author in the field with 35 diverse human brain atlases created, where 15 of them have been released for the global use by Thieme Medical Publishers. Making an overview of a field also encourages an attempt to predict future developments in brain atlasing. On one hand, the future brain research directions are well determined in the brain big projects, such as the BRAIN Initiative that sets six grand goals (BRAIN Working Group 2014). These efforts will result in the acquisition of more and more massive amounts of data and the creation of more advanced and complex brain atlases with an ever-growing scope, population, and spatial and temporal resolutions, additionally empowered by more advanced tools. On the other hand these efforts keep increasing a sort of atlas landscape inhomogeneity as well as difficulty in the atlas standardization and the integration and interpretation of various outcomes. Moreover, as the majority of efforts is devoted to brain atlas-related research, we can expect a growing imbalance and chasms among research, clinical, and educational applications of human brain atlases. There are at least three central components related to atlas standardization, namely, an atlas coordinate system, a core reference cerebral model, and a brain atlas platform architecture. The two most widely used coordinate systems in the neuroscience community are the Talairach system (Talairach and Tournoux 1988) and the Montreal Neurological Institute (MNI) system, and any coordinates of the latter can be converted to the Talairach space (Chau and McIntosh 2005) . The Talairach coordinate system has become the standard reference for reporting the brain locations in scientific publications, though its definition is not unique. The Talairach system is based on the anterior (AC) and posterior (PC) commissure line and its center is located at the AC point landmark. However, the AC and PC point landmarks can be defined at least in four different ways resulting in a substantial discrepancy among the coordinates depending on a selected landmark definition (Nowinski 2001b) . Typically the centers of the AC and PC structures are taken as the point landmarks, while the originally defined point landmarks by Talairach are beyond the AC and PC structures (consequently, for instance, the AC is missing on the coronal plane passing through the center of the coordinate system (and in my print version of his atlas, prof. Talairach "corrected" that by manually drawing it)). A core reference high-quality cerebral model is missing in neuroinformatics. An example of such a longlasting reference model for the cerebral cortex are Brodmann's areas (Brodmann 1909 ). Brodmann's areas, though being one century old and based on a single brain specimen, are most widely used and remain until today applicable references in human brain mapping to correlate functional activations to the underlying neuroanatomy (Amunts and Zilles 2015) , despite the creation of more advanced and accurate cortical maps (Glasser et al. 2016) . For a certain period the Talairach and Tournoux (1988) atlas has played a similar role for the whole brain, despite its well-known limitations including spatial consistency as quantified by Nowinski and Thirunavuukarasuu (2009) . Another example is the Schaltenbrand and Wahren (1977) atlas that for a few decades until the present remains the reference in stereotactic and functional neurosurgery. The construction of the core virtual brain model of the highest possible quality is a complicated, tedious, and timeconsuming process, which requires sophisticated, dedicated, and precise tools and, of course, the state-of-the-art data, besides meticulous attention to details. Therefore, such a virtual brain model shall be built incrementally. We have attempted to create this kind of virtual brain model from multi-modal, multi-sequence scans of a living specimen (in a process which took almost 15 years until funding lasted), see Fig. 2 . This model has been designed as modular (Nowinski 2017b ) with its consecutive modules being developed and validated (including cortical areas and subcortical structures (Nowinski et al. 2012a) , white matter tracts (Nowinski et al. 2012b ), intracranial vasculature (Nowinski et al. 2011a) , cranial nerves and nuclei (2012c), head muscle and glands (2013c), extracranial vasculature (2015b), skull (2015c), and systems, while releasing subsequent five versions (termed The Human Brain in 1492/1969/2953 Pieces) for public use (Nowinski et al. 2011b; 2015a; Nowinski et Chua 2014) . Our effort, though uncompleted, has demonstrated the feasibility of this approach. Although the advantages of population atlases are enormous and obvious, we believe that these atlases shall be constructed around a very detailed, accurate, fully segmented, completely labeled, validated, and deterministic core model of a virtual brain created with the highest quality possible and accepted as the reference standard (similarly as Brodmann created his long-lasting standard for the cortical areas from a single specimen). Moreover, the use of a single specimen enables its continuous rescanning to create new modules with advances in imaging technology (for instance, to build our virtual brain model, the same specimen was rescanned for over 10 years on various 1.5T, 3T, and 7T as well as CT and US (ultrasonography) scanners). Creating a population brain atlas even with a high number of specimens but without ensuring the highest quality and thorough validation just increases the abovementioned inhomogeneity of the field with a difficulty to cross-relate various atlases. The final factor that might potentially counterbalance this atlas inhomogeneity trend is the establishing a standardized, general architecture of the human brain atlas platform supporting equally research, clinical, and educational applications and enabling the clinicians to grow the initial core brain model with their own new data. We believe that the future human brain atlas-related research and development activities shall be founded on and benefitted from such a standard framework containing the core virtual brain model cum the brain atlas platform general architecture.
Summary We have attempted here to track enormous transformational advances in the human brain atlas evolution from hand-drawn cortical maps to print brain atlases to digital atlases with tools to multi-modal and population atlases in health and disease to mega multi-atlases across the lifespan to atlas platforms at macro, meso, micro, and nanoscales, as diagrammatically summarized in Fig. 1 . This atlas evolution review differentiates from other works, usually focusing on the atlas content mostly in research applications, as we take here a wider perspective and analyze this evolution in four categories: content, applications, functionality, and availability. Four generations of human brain atlases are distinguished, namely, early cortical maps, print stereotactic atlases, early digital atlases, and advanced brain atlas platforms. The development of electronic brain atlases spans the last two generations and in this area we identify five avenues, the recent and most prominent is the creation of new electronic brain atlases. The brain atlas content evolution in this avenue is categorized in eight groups taking into account scope, parcellation, modality, plurality, scale, ethnicity, abnormality, and a mixture of them, in which, in turn, the atlas developments are heading in 23 various directions. We suggest that the future human brain atlas-related research and development activities shall be founded on and benefitted from a standard framework containing the core virtual brain model cum the brain atlas platform general architecture. 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/. Fig. 2 The virtual decomposable brain model extended to the head and neck with about 3,000 fully segmented, labeled, and color-coded 3D components. Shown here: the right central nervous system with the cerebrum (parcellated into gyri and sulci), cerebellum, brainstem, and cervical spine; deep gray nuclei; cerebral ventricles; white matter (deep and posterior fossa); white matter tracts; right visual system; auditory system; intracranial arteries; intracranial veins; dural sinuses; cranial nerves with nuclei (partly exposed on the left side); right head (masticatory) muscles; right glands; upper skull with the frontal bone removed; cervical spine (3rd and 4th cervical vertebrae); extracranial arteries; and extracranial veins
; de Haan and Karnath 2017). 8. Multiple (combined) groups a. Population multi-modal atlases (Iglesias et al. 2018); b. Population functional maps and atlases (Nowinski et al. 2003; Nowinski 2009; Breshears et al. 2015); c. Population spatio-temporal atlases, for instance, of brain development (Kuklisova-Murgasova, et al. 2011); d. Population ethnic atlases and templates, for instance, Chinese brain atlas (Tang et al. 2010), Indian brain template (Bhalerao et al. 2018) and atlas (Sivaswamy et al. 2019), Korean brain template (Lee et al. 2005), and French brain template
Table 1 1 Number of citations under the term Year Number Year Number "human brain atlas" on PubMed versus years of 1950 1 2011 174 publications (as of 18 1960 1 2012 213 May 2020) 1970 3 2013 241 1980 5 2014 297 1985 18 2015 312 1990 16 2016 337 1995 22 2017 362 2000 42 2018 474 2005 121 2019 389 2010 185 2020 132
Note that we were the only research group that received rights from the publisher to use these atlases.
Neuroinform (2021) 19:1-22
|
10.1186/s00015-022-00429-6
|
cc-by
| null | null |
openalex
|
The Schistes Lustrés form a large and complex unit at the top of the Penninic nappe stack of the Alpine belt. Calcschists, partly of Late Cretaceous age, constitute the dominant lithology. They are closely associated both with blueschist facies Piemont-Ligurian ophiolites and continent-derived Mesozoic metasediments. The question of whether the Schistes Lustrés originated on continental or oceanic crust has been extensively debated among Alpine geologists and is locally still controversial. We present here new structural and stratigraphic observations, as well as Raman graphite thermometry (RSCM) data, for the Schistes Lustrés complex of the Combin zone in the Hérens, Dix and Bagnes valleys. Our observations indicate that the basal part of this Schistes Lustrés complex (defined as the Série Rousse) is systematically devoid of ophiolitic material, and rests in stratigraphic contact on the underlying Triassic -Lower Cretaceous metasediments and Paleozoic basement of the Mont Fort nappe (Prepiemont paleogeographic domain). The unconformity at the base of the Schistes Lustrés complex is interpreted as resulting from the sedimentation of the Série Rousse on a paleorelief formed by remnants of Jurassic normal fault scarps, and not as an Alpine tectonic contact, as previously proposed. The lithostratigraphic comparison with the Breccia nappe in the Prealps, as well as a foraminifer discovery, allows us to better constrain the age of the Série Rousse. It extends from the middle of the Early Cretaceous (Aptian?) to the Late Cretaceous (Campanian to earliest Maastrichtian?). In contrast, the upper contact of the Série Rousse with the ophiolite-bearing Schistes Lustrés clearly corresponds to an Alpine thrust. The thrust zone is underlined by thin and discontinuous slices of highly strained continental-margin derived Mesozoic metasediments (Frilihorn slices). RSCM data show that the recrystallization of the organic matter progressively increases on both sides towards this contact. This contact, internal to the Schistes Lustrés complex, is reinterpreted as the major tectonic contact separating the Middle Penninic Mont Fort nappe from the Upper Penninic Tsaté nappe (defined here as including only the ophiolite-bearing Schistes Lustrés and associated meta(ultra-)basites). This study clearly documents that the Schistes Lustrés consist of sediments either deposited on oceanic crust, showing locally Editorial handling: Paola ManzottiIntroduction The Schistes Lustrés form one of the largest lithological groups in the Western Alps and Western Central Alps (Fig. 1 ). They crop out in the upper part of the Penninic nappe stack in an intermediate tectonic position between the units derived from the Briançonnais (s.l.) continental margin, the eclogitic Piemont-Liguria ophiolites and the units derived from the Adriatic continental margin (Fig. 1 ). They constitute a set dominated by calcschists showing ophiolitic intercalations, up to several hundred meters thick. The name Schistes Lustrés refers to the glossy metallic grey ("lustré") aspect of these calcschists. It is the abundance of phyllosilicates and graphite, which gives, together with the metamorphism, this characteristic aspect to their schistosity surfaces. The question of the origin of the Schistes Lustrés has always been problematic, notably because of their close association with metabasites and mantle rocks as well as with continental margin successions. The extreme scarcity of fossils also makes the determination of their age difficult (e.g. Termier 1902; Staub 1942a ). Since the emergence of plate tectonics, the question of the continental or oceanic nature of the bedrock on which the Schistes Lustrés were deposited has been extensively debated among Alpine geologists. The idea that both cases could exist, or even coexist, appeared early on and have been proposed by different authors (e.g. Staub 1942b; Lemoine 1953 Lemoine , 1964 Lemoine , 1967 Lemoine , 1971;; Elter 1971; Bearth 1976; Bourbon et al. 1979; Caby 1981; Lemoine and Tricart 1986; Fudral et al. 1987; Lagabrielle 1987) . However, in several cases the question still remains relevant today as the stratigraphic or tectonic nature of the contact between the Schistes Lustrés and the surrounding units is most often difficult to establish (Lemoine 1971; Michard and Schumacher 1973; Marthaler and Stampfli 1989) . Indeed, a strong ductility contrast generally characterizes these interfaces and favors their late movements in the form of local shearing. The difficulties also result from the strong lithological similarities between the Schistes Lustrés of oceanic origin and those originating from continental margins. The contact between such units may be difficult to identify, especially since they are now involved in nappes and have undergone polyphase ductile deformation (e.g. Savary and Schneider 1983) . The present study focuses on the Schistes Lustrés of the Combin zone (Pennine Alps), in the area located NW of the Dent Blanche klippe (Fig. 1b ; Canton of Valais, Switzerland).
Geological setting
The Schistes Lustrés in the nappe stack of the Pennine Alps The metasediments and ophiolitic remnants constituting the Schistes Lustrés complex originated from the Mesozoic Piemont-Liguria basin and its margins. This basin, which constituted the main branch of the Alpine Tethys, was a slow-spreading small oceanic domain, resulting from the rifting between the European and Adriatic plates (e.g. Lemoine and Trümpy 1987; Lagabrielle and Lemoine 1997; Stampfli et al. 2002; Le Breton et al. 2021; Manatschal et al. 2022) . Oceanization of the basin is attested since the Bajocian (Elter et al. 1966; Bill et al. 2001; Manatschal and Müntener 2009 ) and continued at least until the Kimmeridgian (e.g. Bill et al. 1997; Rubatto et al. 1998; Schaltegger et al. 2002; Decrausaz et al. 2021) . The progressive closure of the Alpine Tethys starting in the Late Cretaceous (e.g. Caron et al. 1989; Gasinski et al. 1997; Stampfli et al. 1998; Skora et al. 2009; Rubatto et al. 2011) , led to the accretion and incorporation of ophiolitic slivers and associated oceanic sediments into the orogenic prism (e.g. Lagabrielle 1987; Marthaler and Stampfli 1989; Stampfli and Marthaler 1990; Stampfli et al. 1998) , and finally to the Cenozoic Alpine collision (e.g. Escher and Beaumont 1997; Schmid et al. 2017; Candioti et al. 2021 ). In the nappe stack of the Pennine Alps, the ophiolitic units derived from the Piemont-Liguria basin show two types of contrasting tectono-metamorphic evolution (e.g. Kienast 1973; Dal Piaz 1974; Ernst and Dal Piaz 1978; Merle and Ballèvre 1992; Ballèvre and Merle 1993; Negro et al. 2013 ). (i) The structurally lower ophiolitic units (Zermatt-Saas Fee, Antrona, and Lanzo) show eclogite facies paragenesis (e.g. Bearth 1967; Pfeifer et al. 1989; Bucher et al. 2005 Bucher et al. , 2019;; Angiboust et al. 2009; Dragovic et al. 2020) with local UHP relics in Zermatt-Saas Fee (e.g. Reinecke 1991 Reinecke , 1998;; Forster et al. 2004; Frezzotti et al. 2011 Frezzotti et al. , 2014;; Groppo et al. 2009 ) and a predominance of ophiolites over metasediments. (ii) The structurally upper ophiolite-bearing unit, the Tsaté nappe (Sartori 1987; Escher 1988; Marthaler and Stampfli 1989) , or Combin zone s.str. (e.g. Dal Piaz 1971; Bearth 1976; Caby 1981) , corresponds to the Schistes Lustrés in a hyper-extended continental margin setting Schistes Lustrés complex. It is dominated by metasediments and shows greenschist facies paragenesis with blueschist-facies relics (e.g. Caby 1981; Bousquet et al. 2004; Manzotti et al. 2021; cf. chap. 2.3.1) . The lithologies and metamorphic paragenesis of (ii) are similar to those of the Schistes Lustrés of the French Western Alps (e.g. Agard et al. 2001; Plunder et al. 2012) . At its top, the Schistes Lustrés complex of the Combin zone is in tectonic contact with the Paleozoic basement of the Sesia and Dent Blanche nappes (Adriatic margin; e.g. Manzotti et al. 2014 Manzotti et al. , 2017;; Angiboust et al. 2014; Kirst 2017; Kirst and Leiss 2017) . At its base, it rests on different tectonic units (Fig. 1): (1) the Zermatt-Saas Fee nappe (Piemont domain), in the internal part of the belt (Ballèvre et al. 1986; Bucher et al. 2004; Dal Piaz et al. 2015) ; (2) the Siviez-Mischabel nappe (Briançonnais domain), north of Zermatt, as well as in the Turtmann and Anniviers valleys and on the right side of the Hérens valley (e.g. Hermann 1913; Bearth 1953 Bearth , 1978;; Sartori 1987 Sartori , 1990;; Scheiber et al. 2013 ); (3) the Evolène Series, cover of the Mont Fort nappe (Prepiemont domain ; Escher 1988; Marthaler 1984; Pantet et al. 2020) , NW of the Dent Blanche klippe; or (4) directly on the Paleozoic basement of the Mont Fort nappe, if the Evolène Series is absent.
Subdivisions in the Schistes Lustrés complex of the Combin zone After the first definition of the Combin zone by Argand (1909) , as a composite tectonic unit grouping together all the Mesozoic metasediments outcropping between the basements of the Grand St-Bernard and Dent Blanche nappes, more specific studies of its Schistes Lustrés complex (Combin zone s.str.) were carried out, for example by Staub (1942a Staub ( , 1942b Staub ( , 1942c)) , Witzig (1948) , Zimmermann (1955 ), Dal Piaz (1965 , 1971) , Bearth (1967 Bearth ( , 1978) ) and Caby (1981) . Further detailed studies of these Schistes Lustrés, in the area located north of the Dent Blanche klippe, allowed to individualize different lithological and tectonic units within this complex (Marthaler and Escher in Masson et al. 1980; Marthaler 1981 Marthaler , 1984;; Escher and Masson 1984; Sartori 1987; Escher 1988; Escher et al. 1988 ; Fig. 2 ). We describe them below, from top to bottom. 1. The upper unit is mainly formed of basic and ultrabasic rock bodies reaching pluri-km sizes, associated with calcschists. This unit corresponds with the Tracuit zone (Zimmermann 1955; Escher and Masson 1984; Sartori 1987) , defined in the eastern part of the study area, and with the ophiolites and associated metasediments of the Aiguilles Rouges d' Arolla (Aiguilles Rouges-Zone and Hochpenninikum höhern Schuppen, Witzig 1948; Série ophiolitique s.s., Kunz 1988 ; Aiguilles Rouges and Mont de l'Etoile Ophiolites, Decrausaz et al. 2021 ). In the following, we will use the term Tracuit -Aiguilles Rouges zone to refer to this unit. 2. The intermediate Schistes Lustrés unit is usually designated by the term Série Grise (Marthaler 1984) . It is composed mostly of calcschists and other oceanic metasediments, mixed with ophiolitic lenses, which are usually smaller than those of the previous unit. 3. A thin unit of Mesozoic metasediments derived from a continental margin (0.1-20 m thick) punctuates the base of the Série Grise as discontinuous and often A. Pantet et al. Fig. 1 (See legend on previous page.) Schistes Lustrés in a hyper-extended continental margin setting strongly tectonized slices. It constitutes the upper and outer digitation of the Faisceau Vermiculaire of Argand (1916a Argand ( , 1916b;; Escher and Masson 1984) . North of the Dent Blanche klippe, its thickness is often below one meter and its maximum thickness (20 m) is reached at the Frilihorn (Fig. 1b ), the summit that gave its name to this unit (Hermann 1913; Marthaler 1984; Sartori and Marthaler 1994; Stampfli and Marthaler 1990 ). 4. The lower Schistes Lustrés unit, called the Série Rousse (Marthaler and Escher in Masson et al. 1980; Marthaler 1981) , consists mainly of calcitic marbles rich in detrital material and of calcschists. The Schistes Lustrés of this unit show a near continental margin affinity contrasting with that of the upper units, reflected in particular by the local presence of breccia levels with dolomitic clasts and by a marked quartzitic detrital component. In the original definition of the Tsaté nappe (Sartori 1987), only the two upper units above, i.e. the Série Grise and the Tracuit (-Aiguilles Rouges) zone, were included in this nappe (Fig. 2 ). The two lower units (Série Rousse and Frilihorn) were attributed to the underlying Mont Fort nappe (e.g. Escher 1988; Escher et al. 1988; Allimann 1990; Deville et al. 1992) . Marthaler and Stampfli (1989) and Stampfli and Marthaler (1990) evidenced the similarities between the Tsaté nappe and modern accretionary prisms, both showing a structure composed of superimposed slices involving sediments and ophiolites. These similarities, together with the identification of large-scale unconformities at the base of the Série Rousse, led to propose the inclusion of the Série Rousse as a basal slice to the Tsaté nappe and to reinterpret the nature of the basal contact of these series as tectonic (Escher et al. 1993; Sartori and Marthaler 1994) . Further evidence for local fluid circulation along the basal contact of the Série Rousse was highlighted by an isotopic profile carried out across this contact (Kramar 1997) . This evidence reinforced the hypothesis of the affiliation of the Série Rousse to the Tsaté nappe, since then accepted and adopted by all subsequent authors (e.g. Escher et al. 1997; Steck et al. 1999; Tectonic map of Switzerland 2005) . According to this redefinition, the Tsaté nappe would correspond to a stack of tectonic slices, grouping together all the Schistes Lustrés of the Combin zone (Fig. 2 ). The different lithologies constituting the Schistes Lustrés north of the Dent-Blanche klippe are described in the following chapter. It gathers the necessary elements that allow the distinction of the different series or units defined in the literature. The new data are based on Fig. 2 Tectonic attributions of the studied units/series between successive previous authors and this study detailed mapping and tectonostratigraphic observations of key areas.
Lithologies of the Schistes Lustrés complex of the Combin zone
Ophiolite-bearing Schistes Lustrés and associated lithologies We will describe the different lithologies of the ophiolite-bearing Schistes Lustrés of the Combin zone independently of the tectonic subdivisions detailed in the previous chapter, because the same lithologies are present in these different subdivisions. The ophiolitic material can be incorporated in these Schistes Lustrés as levels or lenses of variable sizes, or as a sand-sized or thinner detrital component.
Calcschists The calcschists of the ophiolitebearing Schistes Lustrés of the Combin zone generally display a greyish and sometimes greenish tint, which may turn to russet for the levels that are the richest in calcite (Fig. 3a ). The characteristic metallic gray appearance of their schistosity surfaces is due to the abundance of phyllosilicates and graphite. Their mineralogy is generally composed of calcite, quartz, muscovite, chlorite, albite and pyrite, ± tourmaline, apatite, ankerite, zircon, rutile, pyrite and various oxides (e.g. Marthaler et al. 2008b Marthaler et al. , 2020b)) . Metamorphic parageneses are mostly typical of greenschist facies, but early blueschist facies parageneses are attested by the local preservation of relics of garnets, Mg-chloritoid and phengite (Burri et al. 1999; Bousquet et al. 2004 Bousquet et al. , 2008)) , of carpholite pseudomorphs (Pfeifer et al. 1991) ; and of lawsonite pseudomorphs and aragonite inclusions in titanite (Manzotti et al. 2021) . The local presence of fuchsite (Vogel 1995; Manzotti et al. 2021) reflects the presence of chromium in these sediments and seems to confirm their oceanic origin. Lithologies are variable and can evolve towards phyllitic and quartzitic marbles, as well as towards black shales, which are often compared to the Palombini shales from the Apennines (e.g. Marthaler and Stampfli 1989 ). In the Tsaté nappe, such dark shales and associated calcschists have been grouped together as the Garda Bordon Formation, which is supposedly of Early to "mid"-Cretaceous age (Marthaler et al. 2020b) . Rhythmic alternations of shales and decimetric beds of quartzitic calcarenites, locally showing graded bedding, have been, for their part, grouped together as the Fêta d' Août Formation (Viredaz 1979; Marthaler et al. 2020b ). This formation is attributed to the Early Cretaceous by analogy with the Replatte formation of the Western Alps (Lemoine and Tricart 1986) . Relics of planktic foraminifera have been described in several localities of the Série Grise (Marthaler 1981 (Marthaler , 1984;; and unpublished diploma theses from: Savary 1982; Schneider 1982; Besson 1986; Du Bois and Looser 1987; Kunz 1988) . They generally come from the levels that are the richest in calcite in this unit. The strong rheological contrast between these levels and the surrounding calcschists, as well as a diagenetic replacement as Fe-carbonate (usually referred to as ankerite), seem to have allowed a local preservation of these forms, despite the intensity of deformation and recrystallization. The forms described mostly recall Rotalipora sp., or more rarely Marginotruncana sp., corresponding to Cenomanian and Turonian-Santonian age ranges, respectively (e.g. Peryt et al. 2022) . Forms of the same types have also been described in various localities of the Schistes Lustrés of the Western Alps (Lemoine et al. 1984; Marthaler et al. 1986; Deville 1987; Fudral et al. 1987) and Corsica (Meresse et al. 2012 ).
Volcanosedimentary arkoses Volcanosedimentary levels are found in several locations in the Schistes Lustrés of the Combin zone (e.g. Kunz 1988; Decrausaz et al. 2021; Fig. 3b) . Kunz (1988) describes in particular volcanosedimentary arkoses at the contact between the ophiolite bodies intercalated in the Schistes Lustrés and the overlying calcschists and black shales. The gradual transition between these lithotypes seems to indicate an original stratigraphic contact of the sediments on their ophiolitic bedrock. Volcanosedimentary levels are also described in the Schistes Lustrés of the Western Alps (e.g. Lagabrielle and Polino 1985) , in particular gabbroic arenites (Le Mer et al. 1986 ) and basaltic sands (Lagabrielle 1987 ).
Calcitic marble levels Several meters to tens of meters thick levels of calcitic marbles and breccias are observed in the ophiolite-bearing Schistes Lustrés of the Combin zone (Staub 1942c; Burri et al. 1998 Burri et al. , 1999;; Marthaler et al. 2020b) . Marbles containing serpentinite clasts, and sometimes abundant epidote, are locally found in stratigraphic contact with ophicarbonates and ophiolitic lenses (Vogel 1995; Marthaler et al. 2020b; Decrausaz et al. 2021; Fig. 3c, d) . Such levels are also described in the Schistes Lustrés of the Western Alps and Corsica (e.g. Lemoine et al. 1970; Lagabrielle et al. 2015) . Their strong analogies with limestones containing ophiolitic clasts of the Upper Penninics of Graubünden in which Weissert (1975) observed Calpionella, and with the Calpionella limestones of the supra-ophiolitic sediments of the Apennines and Corsica (Abbate and Sagri 1970; Andri and Fanucci 1973) , makes their attribution to the latest Jurassic / earliest Cretaceous very likely. larites, are intercalated in the Schistes Lustrés of the Combin zone (Staub 1942c; Hagen 1948; Witzig 1948; Bearth and Schwandler 1981; Caby 1981; Ayrton et al. 1982; Marthaler 1984; Burri et al. 1999; Marthaler et al. 2020b) .
Manganese-bearing quartz micaschist beds Spessartine is common and is sometimes accompanied by other manganese minerals or mineralizations (e.g. Baldelli et al. 1983; Ansermet and Meisser 2012; Marthaler et al. 2020b) . Piemontite, accompanied by braunite and Mngarnets, have been observed in such levels near the Chanrion hut in the Bagnes valley (Burri et al. 1999; Fig. 3e Dal Piaz 1971; Kunz 1988; Decrausaz et al. 2021 ). Basaltic breccias have been described, particularly at the contact with the underlying ultramafic rocks (e.g. Caby 1981) . The metagabbros can form plurikilometric bodies, as for example in the region of the Tracuit alp; and at the Aiguilles Rouges d' Arolla (Fig. 1b ), where they have been dated at 154.9 ± 2.6 Ma and 155.5 ± 2.8 Ma (U-Pb on zircon, LA-ICP-MS; Decrausaz et al. 2021) . The most common ultrabasites in the Combin zone are serpentinites. They are intercalated as lenses that can reach plurikilometric dimensions. Their mineralogy is dominated by antigorite, generally accompanied by magnetite and tremolite (Angiboust et al. 2014) . The borders of the lenses can be enriched with talc, antigorite, magnesite and dolomite (Pfeifer and Serneels 1988; Angiboust et al. 2014) . Serpentinite breccias can be observed locally at the top of these lenses, in contact with the metasediments, and probably result from continued Jurassic rifting (Vogel 1995; Decrausaz et al. 2021; Fig. 3f ).
Frilihorn Series In the study area, north of the Dent Blanche klippe, the thin and discontinuous Frilihorn Series is mostly reduced to a few tens of cm of strongly tectonized carbonate rocks. Where the series are the thickest and least deformed, different types of Mesozoic metasediments can be recognized (e.g. Hermann 1913; Witzig 1948; Marthaler 1984; Allimann 1990; Sartori 1990 ). Dolomites constitute the dominant lithology. They are observed in particular at the Bail de l' Ardzentière [2′594′200/1′093′000] in the Bagnes valley (Besson 1986) and NE of Molignon [2′605′540/1′104′600] in the Hérens valley. Calcitic marbles are also common, they are generally clear-colored, relatively pure and form notably the 15 m cliff of the summit of the Frilihorn (Marthaler 1984) . Thin layers of dolomitic breccias intercalated within this level can be observed there. This same level can be followed through the Arpettes pass towards the Turtmann glacier (cf. Tectonic panorama from Hermann 1913), which it crosses at an altitude of 2700 m, until the NE face of the Adlerflüe, where it forms a clearly visible 10 m thick band of white calcitic marbles. There, a level of breccia, already indicated by Sartori (1990) , is present at its base. The mm-to cm-sized clasts are mostly dolomitic and the matrix consists of a dark calcitic marble. Similar breccias are also observed SE of L' Ata Gieute [2′604′625/1′104′755] in the Hérens valley, where they surround a 3 m thick dolomitic level. In the study area, quartzites are very rare in this unit. A 20 cm thick level of micaceous quartzite is nevertheless present near the top of the Frilihorn, intercalated between the marbles of the upper cliffs and russet calcschists covering the summit itself. These calcschists, which contain dolomitic clasts at their base (Marthaler 1984) , as well as similar calcschists and russet phyllitic marbles from a few other outcrops, are also attributed to the Frilihorn Series (Marthaler 1984; Sartori 1987; Allimann 1990 ).
Série Rousse The Série Rousse consists of calcschists, phyllitic and quartzitic marbles, dark shales and thin conglomeratic layers, forming the base of the Schistes Lustrés complex of the Combin zone (e.g. Marthaler 1984; Marthaler et al. 2020b) . Its thickness can reach several hundred meters. The calcschists of these series are generally poorer in phyllosilicates than those of the ophiolite-bearing Schistes Lustrés, whereas the levels of calcitic marbles are more abundant and thicker. The name Série Rousse (Marthaler and Escher in Masson et al. 1980 ) comes from the frequent russet colored patina, typical of the sediments of these series (Fig. 4a-c ). It is due to the presence of iron oxides, associated with the detrital quartz content, and iron carbonates (e.g. ankerite) in the carbonaceous levels. The tint of the patina contrasts with the glossy metallic gray tint of the schistosity surfaces resulting from the abundance of graphite and white micas in these sediments (Fig. 4a ). The mineralogy is typically composed of calcite, quartz, white micas, albite, graphite and pyrite (often idiomorphic) ± chlorite, titanite and zircons. Clasts, mostly dolomitic (Fig. 4c, d ), appear in some levels (Marthaler et al. 2008b (Marthaler et al. , 2020b)) . Garnet relics and pseudomorphs after lawsonite can be observed locally (Besson 1986; P. Manzotti pers. com.) . Relics of planktic foraminifera, diagenetically transformed into Fe-Mg carbonates, have been described in several locations in the Série Rousse (Marthaler 1981 (Marthaler , 1984 ; and unpublished diploma theses from: Pilloud and Sartori 1981; Schneider 1982; Crespo 1984; Besson 1986; Schmid 1988; Salamin 1989 ). The described micropaleontologic content presents forms that essentially ascribe to Rotalipora sp. (of Cenomanian-type), with a few bicarinate forms indicating a Turonian to "early Senonian" (i.e. Coniacian-Santonian) age (Marthaler 1981 (Marthaler , 1984)) , for the younger parts of the series.
Methods
Field observations and mapping This study was based essentially on an extensive geological field work conducted over five consecutive seasons. A relatively large-scale approach was favored (studied area extending through the Bagnes, Dix, Hérens, Anniviers and Turtmann valleys; Fig. 1b ), while the level of detail of the observations was adapted to the interest and quality of the outcrops.
Cathodoluminescence (CL) Cathodoluminescence, or the emission of photons in the visible range of the electromagnetic spectrum under cathodic excitation, is a technique routinely used in mineralogy and sedimentary petrography, as it may enable to distinguish patterns of trace elements remaining undetectable under classical optical microscopy. CL depends on the presence of activator ions, which are stimulated to emit light when bombarded with energetic electrons, or on the presence of quencher ions. In carbonates, the best-known activator is Mn 2+ . Bivalent iron (Fe 2+ ) is the most important quencher ion (e.g. Machel et al. 1991) . In metasediments showing advanced calcite recrystallization, CL can allow to highlight the presence of microfossils or to distinguish sedimentary structures, no longer recognizable under classical optical microscopy. CL images were obtained at the University of Lausanne using a Technosyn 8200 MkII mounted on an Olympus BH-2 microscope, and operated at 15-20 kV and 0.4-0.5 mA with an unfocused cold cathode electron beam under a He atmosphere at 0.2 torr.
Raman spectroscopy of carbonaceous material (RSCM) Raman graphite thermometry is based on the progressive transformation of organic matter into graphite with increasing temperature. As this transformation is considered to be irreversible, the structural organization of the carbonaceous material (CM) records the maximum temperature reached during metamorphism (Wopenka and Pasteris 1993; Beyssac et al. 2002 Beyssac et al. , 2003a)) . Beyssac et al. (2002) obtained a linear correlation between the peak temperature and the structural organization of CM (RSCM method). This method enables the determination of the peak temperature in the range of 330-650 C with an absolute accuracy of ± 50 C. Relative uncertainties on peak temperature may however be much smaller (e.g. Beyssac et al. 2004; Wiederkehr et al. 2011; Negro et al. 2013; Angiboust et al. 2014) . Raman spectroscopy was performed at the University of Lausanne using a HR Raman-FTIR spectrometer from HORIBA Scientific, an integrated Raman microprobe consisting of an Olympus BX41 confocal microscope coupled to an 800 mm focal-length spectrograph. A 532.12 nm frequency doubled Nd-YAG continuous wave laser was focused on the sample. The power of the laser at the surface of the sample was 9 mW. Analytical procedures from Beyssac et al. (2002 Beyssac et al. ( , 2003b) ) were followed closely while also taking into account the recommendations reported in Beyssac and Lazzeri (2012) : measurements were carried out on polished thin sections, and CM was systematically analyzed below a transparent adjacent mineral, most often quartz. The sampled volume was a few μm 3 using a 100 × objective. The Raman signal was collected in backscattered mode. Analyses were carried out using a grating of 1800 grooves/mm, a 150 μm slit aperture and a 150 μm hole. Spectra were recorded in extended scanning mode (1100-1800 cm -1 ) using the LabspecTM v.4.15 software and an acquisition time of 2 × 40 s. The spectrometer was calibrated with a silicon standard before each session. Spectra were acquired in 12-35 different spots on each sample. They were processed using the PeakFit v4.12 software. A linear baseline correction was applied using systematically the same parameters (2nd derivative zero linear correction, tolerance 0.5%, zero negative correction). Peaks D1 (~ 1350 cm -1 ), G (~ 1510 cm -1 ) and D2 (~ 1620 cm -1 ) were systematically fitted, peak D3 (~ 1510 cm -1 ) was fitted additionally when present. Spectra which could not be fitted with r 2 coef. >= 0.995 (PeakFit variance analysis), were rejected. For each sample, temperatures calculated from 10 to 16 spectra (using equations from Beyssac et al. 2002) were averaged to obtain the peak temperature value.
New lithological observations
Série Rousse
Lithological descriptions and successions
Series devoid of ophiolitic material Following our observations, the Série Rousse is systematically devoid of ophiolitic material. A metric level of prasinites, intercalated in the calcschists between the SE side of the village of Evolène and the vicinity of the Sasseneire summit, has however been assigned to these series by some authors (Allimann 1987 (Allimann , 1990;; Marthaler et al. 2020b ) and would represent the only ophiolitic occurrence attributed to these series. According to our interpretation, these ophiolite-bearing calcschists do not belong to the Série Rousse, but to the overlying Série Grise, forming a pinched syncline in this area (Fig. 1b ). Indeed, associated to these prasinites, ultrabasic rocks can also be observed locally, and in particular, a lens of several meters long of talcschists described by Gerlach (1861), Pfeifer et al. (2011 ), Marthaler et al. (2020b) . Serpentinite lenses of a few cm are associated to the talcschists at point [2′605′040/1′106′260]. Additionally, the calcschists hosting these ophiolitic intercalations are significantly richer in phyllosilicates than those of the surrounding Série Rousse. Our RSCM data (cf. chap. 6) point to the presence of a tectonic contact located between these calcschists and the adjacent Série Rousse.
Thickness of the series The Série Rousse shows large variations in thickness. It reaches several hundred meters in several sectors of the study area (e.g. in the Dix valley), but can be reduced locally to a few tens of meters, as in the thin band bordering the frontal folds of the Mont Fort nappe, east of the village of Evolène (Fig. 1b ). These important thickness reductions can be partly of tectonic origin (strongly stretched fold limbs), but can locally also have a stratigraphic origin (cf. chap. 7.1.2).
Quartz-phyllitic calcitic marbles and calcschists At the base of the Série Rousse, the lithologies are frequently massive and contain less micas over the first few meters, or sometimes several tens of meters. They generally consist of calcitic, quartzitic and phyllitic marbles. Detrital quartz, which can be very abundant, is either disseminated in the calcitic matrix, or occurs as size-sorted rounded grains of a few tens of hundreds μm size; it is sometimes accompanied by dolomitic grains of arenitic size (Fig. 4d ). These basal lithologies are locally conglomeratic (cf. chap. 5.1.2). The clasts are mostly made of dolomites and limestones (Fig. 4c ), sometimes of quartzite or gneiss; their size is millimetric to centimetric, sometimes metric. The micaceous component increases upwards in the series, which is dominated by calcschists in its upper part. Calcite remains however more abundant in the calcschists of the Série Rousse than in those of the overlying ophiolite-bearing Série Grise. Marthaler et al. 2020b ). These authors note the analogy between these siliceous schists and those of the Joux Verte Fm. from the Breccia nappe in the Chablais Prealps (Dall' Agnolo 1997 , 2000) , dated by planktic foraminifera from the "mid"-Cretaceous (late Barremian -middle Turonian). The Joux Verte Fm. shows different levels of dark pelites, correlated at the scale of the Breccia basin and corresponding, according to Dall' Agnolo (1997 , 2000) , to "mid"-Cretaceous global anoxic events (e.g. Jenkyns 1980; Erbacher and Thurow 1997) . The dark basal levels of the Série Rousse also show microscopic facies analogies with those of the Joux Verte Fm. In particular, dark mottled micrites contain rhombohedral automorph silicates of a few hundred μm size (Fig. 4f ; Raman spectra indicate feldspar), strongly reminiscent of silicified dolomite rhombohedra described by Dall' Agnolo (1997, p. 177) in similar dark micrites containing radiolarians and echinoderm remains. The dark micrites of the base of the Série Rousse also contain locally abundant circular siliceous elements of homogeneous ca. 100 μm size that may correspond to radiolarians and possible echinoderm remains (Fig. 5d, f , g; cf. next paragraph).
Dark calcschists and marbles, black schists (See figure on next page.) Fig. 5 Potential microfauna observed in Série Rousse samples. CL: cathodoluminescence; PPL: plane-polarized light. a Thin section from a conglomerate of the very base of the Série Rousse; Pic d' Artsinol [2′599′170/1′108′585]; sampled a few meters from Fig. 8a . The thickness of the conglomeratic level does not exceed 2 m; the mm-to cm-sized clasts are mainly made of dolomites and limestones; a gradual transition is observed to the overlying typical calcschists of the Série Rousse. In comparison with the intense strain generally observed in other samples of the Série Rousse, strain is locally exceptionally low, which allows the preservation of some microfauna that is generally entirely destructed elsewhere. Rounded borders of the clasts could suggest an incomplete diagenesis of the clasts, before their reworking into the conglomerate. b Holothuroidea fragments from the matrix of the conglomerate in the same thin-section. c Holothuroidea fragment from arenitic calcschist of the Série Rousse, sampled few meters above. d Probable reworked echinoderm remains; shapes of the dark rims (CL) suggest stereomes of crinoid ossicles. e Probable planktic foraminifera observed as intraclast in a rounded, dark micritic limestone pebble. The form may correspond to Globotruncana neotricarinata, Petrizzo, Falzoni and Premoli Silva (2011) indicating an age range from Campanian to earliest Maastrichtian for the rounded clast. In order to preserve the observed form, the polishing protocol was not fully completed on this thin section.
Microfauna description Thin sections of about forty samples of the Série Rousse were examined under the microscope for the detection of microfossil relics. The study under transmitted light (TL) having proven unsuccessful, it was completed by a cathodoluminescence (CL) examination. It allowed the detection of higher Mn 2+ trace element concentrations in microfossil relics than in the surrounding sediment, despite the high degree of recrystallization. This method allowed the identification of different biogenic features within a few samples, that were particularly spared from the intense regional deformation.
Samples from the base of the Série Rousse north of the Pic d'Artsinol Some samples collected at the base of the Série Rousse, close to the summit of the Rionde de Vendes [2′599′170/1′108′585], show little deformation, and hence, relatively well-preserved sedimentary features (Fig. 5a ). The very competent lithologies of these levels and of the underlying polygenic breccias of the Evolène Series, have probably contributed to this unusual preservation. These samples consist of phyllitic and quartzitic calcitic marbles, containing mm-to cm-sized clasts of dolomite, quartz, pelagic limestone and quartzschist. Different biogenic features are observed in the thin section of Fig. 5a . The matrix contains some rare dark (TL and CL) round elements of ± 200 μm size, showing regular round openings filled with carbonate of dull orange CL-emission. We interpret these features as originally pyritized and now oxidized holothurian skeletal elements (Fig. 5b ; a similar form was found in the sample of Fig. 5c , few meters above). In the matrix of the first sample, some dolomitic laminae most probably represent reworked echinoderm remains (Fig. 5d ). Particular shapes of the dark rims (CL) actually suggest stereomes of crinoid ossicles. As both crinoids and holothurians have existed throughout the Mesozoic from shallow shelf to deeper marine environments, they are not age diagnostic, but are, nevertheless, indicative of a higher nutrient environment in their source area, perhaps on an outer slope. Pyrite is preserved in some thin sections and the pyritization of echinoderm remains is common in nutrient-rich paleoenvironments. Some mm-to cm-sized disrupted clasts of pelagic limestone show preserved lamination and organic matter. These clasts show two distinct lithologies in original sedimentary contact, which can be followed across adjacent fragments (Fig. 5a ): a lighter grey micrite; and a dark grey, mottled micrite rich in carbonaceous material (confirmed by Raman). The mottles are in fact flattened flakes that could represent original faecal pellets. The borders of the pelagic limestone clasts are fuzzy, suggesting penecontemporaneous reworking into a pebbly sandstone. In CL, the two pelagic lithologies show uniform mauve colors with some blue specks (disseminated biogenic quartz?) and rare bright yellow-orange lens-shaped structures that could represent fossil remains. The mauve color of the pelagic matrix (e.g. Fig. 5e ) indicates a mixture of the intrinsic blue (± 410 nm) calcite CL, with very low (< 1 ppm) Mn 2+ orange (600 nm) activation, while the possible fossil remains contain > 10 ppm of Mn 2+ . The orange colored intraclast of Fig. 5e shows bright yellow outlines (CL) that strongly suggest a double-keeled planktic foraminifer. This form suggests a low trochospiral test with a convex spiral side and two equidistant keels, parallel to the coiling axis and separated by a narrow band. Chambers of the last whorl are inflated and trapezoidal on the umbilical side, with an apparent third keel. Such a description would correspond to Globotruncana neotricarinata Petrizzo, Falzoni and Premoli Silva (2011) . This species has, according to Petrizzo et al. (2011) , a diachronous first appearance ranging from the late Santonian (Exmouth Plateau, NE Australia) to the base of the Campanian (Bottacione, Umbria, Italia). It disappears within the Gansserina ganseri Zone, which reaches the earliest Maastrichtian. Hence, a Campanian to earliest Maastrichtian age range for the pelagic limestone clasts can be inferred. This age is thus slightly younger than the one deduced from the foraminifera described in the Série Rousse so far (cf. chap. 2.3.3). Unfortunately, this finding cannot be confirmed for now with more material. A Campanian maximum age of the Série Rousse must remain a working hypothesis.
Sample from a micritic limestone clast from the Série Rousse NE of Evolène A sample from a meter-sized clast of micritic limestone, included at the very base of the Série Rousse, SW of the Sasseneire in the Hérens valley [2′606′105/1′109′340], shows in thin section numerous circular features, white in TL, of a homogeneous size of about 100 μm (Fig. 5f, g ). Their color contrasts strongly with the very dark color of the rock (micritic limestone transformed into calcitic marble). The observation of these features in CL allows to highlight chemical variations between their internal part (appearing orange red; Fig. 5f CL ), their border (appearing bright yellow; Fig. 5f, g ) and the surrounding sediment. Their shape, size, distribution in the sediment and the aspect of their borders (appearing fuzzy in TL and discontinuous in CL; Fig. 5f, g ), suggest that these forms correspond to radiolarians. These border aspects could reflect their porous nature and the partial filling of these pores by the surrounding sediment.
Mauvoisin marbles Massive, slightly detrital siliceous calcitic marbles (Fig. 6 ) form the bedrock of the Série Rousse in several sectors of the study area. They are best developed in the area of the Mauvoisin dam, where they reach several hundred meters in thickness (Fig. 7a ). On the 1:25'000 geological map (Burri et al. 1998 (Burri et al. , 1999)) , they are referred to as "unité de Mauvoisin", one of the tectonic units defined in the Tsaté nappe by these authors. We propose to use the term Mauvoisin marbles to designate this lithostratigraphic formation. These characteristic siliceous calcitic marbles can be recognized in other zones of the studied area, in particular in the Hérens valley, where they form massive cliffs, well exposed under the villages of La Sage and Villa. In this zone, the best outcrops can be observed south of the hamlet of La Tour, at that of L' Ata Gieute, at Plan Tsardon [2′603′250/1′105′680], at La Vieille [2′602′680/1′105′660] and at La Noûva [2′600′320/1′107′300; 2′600′260/1′107′840]. The characteristic facies of the Mauvoisin marbles is a massive, quartzitic and slightly phyllitic calcitic marble in decimetric to metric beds, showing millimetric to pluricentimetric thick siliceous bands, often standing out in relief on outcrop surfaces (Fig. 6a-c ). The mineralogy is composed of calcite, quartz, white mica, albite, chlorite, , c graphite and oxides ± epidote, tourmaline, apatite and titanite; in the siliceous levels, quartz can constitute more than 80% of the rock (Burri et al. 1999) . Locally, millimetric to centimetric clasts are present; they are mainly made of dolomites and limestones (Fig. 6d ). In places, these clasts can be relatively abundant and a few levels of breccia, generally matrix supported and sometimes slightly graded, are visible (Fig. 6e ). The siliceous banded calcitic marbles described above may also evolve into very siliceous and massive calcitic marbles in some areas. Although these different levels have sometimes been grouped together with the overlying Schistes Lustrés (e.g. Gouffon and Burri 1997; Burri et al. 1998 Burri et al. , 1999)) , or more specifically with the Série Rousse (Allimann 1990; Steck et al. 1999) , their massive aspect and their calcite-and quartz-rich composition clearly distinguish them from these units (cf. Sartori and Marthaler 1994) . No fossils have been found in the Mauvoisin marbles. Some authors have proposed an Early Jurassic age for part of these levels (Sartori and Marthaler 1994; Burri et al. 1999; Marthaler et al. 2020b ; , by analogy with some facies of the Briançonnais Lower Jurassic. Marthaler et al. (2020b) also note analogies between the siliceous banded calcitic marbles from La Vieille [2′602′680/1′105′660] and lithologies of the Bonave Fm. from the Breccia nappe in the Prealps, which have been dated by Calpionella from latest Jurassic/Early Cretaceous (Dall' Agnolo 1997 , 2000) . Sartori et al. (2006, fig. 18) suggest the same correlation for similar white marbles, overlying the polygenic breccias of the Pic d' Artsinol area. Our observations throughout the study area confirm the clear analogy between the dominant facies of the Mauvoisin marbles (slightly micaceous siliceous marbles with mm-cm thick siliceous bands) and those described in the Bonave Fm. In particular, the following similarities can be noted. (1) Layers of graded breccias, with clast sizes up to a few cm, occur at various levels of the Bonave Fm. (Dall' Agnolo 1997 , 2000) , similar to those observed in the Mauvoisin marbles (Fig. 6e ). (2) In the Mauvoisin marbles, some siliceous bands show sharp external borders and locally a progressive passage towards a less siliceous internal part (Fig. 6f ), arguing for a biogenic origin of silica, diagenetically concentrated in replacement chert bands. They could thus correspond to metamorphic equivalents of the characteristic siliceous levels and cherts of the Bonave Fm. (3) In the upper levels of the Bonave Fm., the cherts and siliceous levels become less abundant and a more diffuse silicification of the limestone is observed, while the detrital component and the frequency of marly interlayers increase (Dall' Agnolo 2000) . A similar facies evolution can be observed, e.g. at Mauvoisin along the road between the hotel and the base of the dam. A transition is observed here from facies showing sharply individualized siliceous bands (Fig. 6a, b ), to massive, almost non-bedded, very siliceous calcitic marbles [2′592′740/1′094′350], that are surrounded by thin layers of micaceous calcschists. The marked similarities between the Mauvoisin marbles and the lithologies of the Bonave Fm. thus suggest a Late Jurassic to Early Cretaceous age for the Mauvoisin marbles. They would therefore constitute the youngest levels of the Evolène Series.
Description of the contacts
Basal contact of the Série Rousse
Basal unconformity In the study area, the Série Rousse systematically overlies the Paleozoic basement of the Mont Fort nappe or its Lower Triassic to Lower Cretaceous autochthonous cover (the Evolène Series; cf. Pantet et al. 2020 ). In the region of Evolène, of the Sasseneire and of Moiry (Fig. 1b ), the Série Rousse envelops the frontal folds of the Evolène Series and forms to the east highly stretched isoclinal folds, which can be followed up to the Turtmann and Matter valleys (Sartori 1987 (Sartori , 1990;; Marthaler et al. 2008a) . The map in Fig. 1b shows that the Série Rousse lies either on the Evolène Series, or alternately directly on the Paleozoic basement of the Mont Fort nappe. This second case is observed in several sectors of the normal limb of this nappe, e.g. in the Col du Vasevay and La Sâle area in the Bagnes valley (Fig. 7 ; Gerlach 1871), in the Lac des Dix area, and in part of its reverse limb (between Evolène and the Montset). Our detailed mapping shows that the Série Rousse actually overlies different levels of the Mont Fort basement and of the Evolène Series (Fig. 7 ); levels whose ages probably range from Ordovician (Gauthiez et al. 2011) to Early Cretaceous (cf. chap. 4.2) . The basal contact of the Série Rousse over the Mont Fort nappe is therefore characterized by a major unconformity.
Deformation along the contact: localized shearing due to rheological contrasts and locally unsheared contact A strong rheological contrast often characterizes the contact between the Série Rousse and the underlying levels of the Mont Fort basement and Evolène Series. Indeed, lithologies constituting the Mont Fort nappe are mostly rheologically strong (e.g. gneisses and metabasites of the Paleozoic basement; breccias, dolomites and quartzites of the Evolène Series), whereas most of the lithologies of the Série Rousse are rich in phyllosilicates and are rheologically weak. These contrasting competences frequently lead to a local shearing of the contact. However, along the sections of the contact where the base of the Série Rousse is itself composed of rheologically strong lithologies (quartzitic marbles, conglomerates), no hint of particular shearing can be detected at the contact (cf. Fig. 8a, e, f ). These observations indicate that the basal contact of the Série Rousse over the Mont Fort nappe does not correspond to a major tectonic contact.
Basal conglomerates A conglomeratic level is locally observed at the base of the Série Rousse. Its thickness can reach a few meters, e.g. west of the Vasevay pass in the Bagnes valley (Figs. 7, 8b) , where it was already mentioned by Hagen (1951) . Most often its thickness is limited to a few tens of centimeters, or a few centimeters only (Fig. 8e, f ). Although these conglomerates can be observed in many localities in the study area (Fig. 8i ), they are most often absent. The matrix of these conglomerates is of the same composition as the overlying calcschists of the Série Rousse. A gradual transition can be observed between the conglomerates and the calcschists, by progressive increase of the proportion of matrix. These conglomerates are generally polymict. The clasts mostly consist of dolomites, limestones and pure or phyllitic quartzites, sometimes of prasinites/ovardites. Locally, clasts made of breccias (dolomitic clasts and calcitic matrix) can also be observed (Fig. 8d ). The presence of these breccia clasts and the abundance of phyllosilicates and detrital quartz in the matrix distinguish these conglomerates from the polymict breccias of the Evolène Series of presumed Late Jurassic age (cf. Pantet et al. 2020) , even if these two formations are sometimes directly in contact (e.g. Fig. 8a ). The discovery of a form probably corresponding to a Globotruncana sp. in a clast of this basal conglomerate of the Série Rousse (cf. chap. 4.1.2), as well as the various presumed relics of Rotalipora sp. and bicarinate planktic foraminifera observed in the matrix of other levels of the Série Rousse (cf. chap. 2.3.3), would indicate a Late Cretaceous age for these conglomerates. In different zones of the study area, a correlation can be established between the nature of the clasts of the basal conglomerate of the Série Rousse and the underlying rocks of the Mont Fort nappe. For example, west of the Vasevay pass (Fig. 7 ), the Série Rousse is in contact with Paleozoic metabasites of the Mont Fort basement (Métailler Fm.) and its basal conglomerate contains here numerous clasts of identical lithology (Fig. 8b, c ). Such a relationship can also be observed near the Col des Roux, where the base of the Série Rousse is locally formed by a conglomerate rich in decimetric clasts of pure quartzites, identical to the Tabular Quartzites of the Evolène Series, underneath the contact (Fig. 8e ). On the other hand, no clast observed in this conglomerate consists of a rock that does not form the Evolène Series or the basement of the Mont Fort nappe (such as e.g. granites or serpentinites). On the contrary, the clasts made of breccias (calcitic matrix, dolomitic clasts), observed in this conglomerate at some localities (e.g. Fig. 8d ), are particularly typical of the Evolène Series. These observations thus argue for a deposition of the Série Rousse in the immediate vicinity of the rocks constituting the present-day Mont Fort nappe. On some outcrops preserved from a too strong deformation, it is possible to note the rounded shape of some clasts of the conglomerate (e.g. Fig. 8f ), which is indicative of rolling of the clasts (mechanical abrasion) and argues for a formation of the conglomerate in a context of stratigraphic onlap on a relief. These different observations thus clearly indicate the stratigraphic nature of the basal contact of the Série Rousse over the Mont Fort nappe.
Basal hardground Strongly oxidized crusts of a few mm thickness are locally observed at the basal contact of the Série Rousse (Fig. 8f , g), on top of Jurassic breccias of the Evolène Series, or of calcitic marbles that would constitute their distal equivalents. Locally, these crusts mostly cover the basal surface of the Série Rousse. We interpret them as corresponding to a hardground. Strongly oxidized mm-to dm-sized clasts are locally observed in the basal conglomerate of the Série Rousse and would correspond to a reworking of such crusts (Fig. 8h ). The presence of this hardground suggests a significant stratigraphic gap and confirms the stratigraphic nature of the basal contact of the Série Rousse.
Upper contact of the Série Rousse with ophiolite-bearing Schistes Lustrés and Frilihorn slices Thin discontinuous tectonized slices of the Frilihorn Series (Triassic-Jurassic/Cretaceous; cf. chap. 2.3.2) frequently mark the contact between the Série Rousse and the overlying ophiolite-bearing Série Grise (Fig. 9ac ). They rarely exceed a few meters in thickness, but can be present along the contact for several kilometers. Such slices are also observed in the same tectonic position, south of the study area, in the Ollomont valley (P. Manzotti and M. Ballèvre, oral communication) and in the Rhèmes valley (Adatte et al. 1992) . Where the Frilihorn slices are absent or hidden by recent deposits, the contact between the Série Rousse and the Série Grise may be difficult to identify. The presence in the calcschists of ophiolitic lenses, of ophiolite-derived detrital material, or locally of fuchsite, sometimes allows the identification of the Série Grise, but the contact is often difficult to follow due to the presence of numerous isoclinal folds (Fig. 9c ). Deformation is generally extremely strong in the vicinity of the contact. It is demonstrated in particular by the intense stretching and boudinage observable in the Frilihorn slices (e.g. Fig. 9a, b ). North of the Dent Blanche klippe, these slices are systematically affected by an intense shearing and are locally reduced to only a few dm-thick tectonized and highly fractured carbonates (e.g. Fig. 9c, d ). An intense shearing is also observable within the calcschists directly surrounding the contact (e.g. Fig. 9e ). The contact is also characterized by the local abundance of quartz veins, that are more than 50 cm thick in several localities (e.g. [2′599′320/1′104′600], [2′601′700/1′105′640], [2′602′800/1′105′510]) and reach a thickness exceeding 1 m at point [2′601′010/1′104′460], SW of the Palantse de la Cretta in the Hérens valley. Our observations thus confirm the tectonic nature of the upper contact of the Série Rousse with the ophiolitebearing Schistes Lustrés, as postulated by all previous authors. The question of the importance of this tectonic contact, in particular whether it corresponds to a major tectonic contact marking a nappe boundary, or even a tectonic domain boundary (Upper/Middle Penninic Domains), as proposed, for example, by Escher (1988) ; or whether it is a contact of lesser importance, internal to the Tsaté nappe as proposed, for example, by Sartori and Marthaler (1994) , will be discussed in chapter 7.3.
Raman graphite thermometry RSCM analyses conducted by Negro et al. (2013) , Angiboust et al. (2014 ), Decrausaz et al. (2021) and Manzotti et al. (2021) reveal significant variations in RSCM temperatures (cf. Beyssac et al. 2002) within the Schistes Lustrés of the Combin zone (Fig. 10c ). In order to better characterize the variability of RSCM temperatures between the different units and series of this Schistes Lustrés complex (cf. chap. 2.2), and in particular between the Série Rousse and the Série Grise, additional samples were analyzed during this study (Table 1 ). Sampling was concentrated on a limited area (Hérens valley sector; Fig. 10a ) in order to avoid possible biases related to regional variations in the intensity of metamorphism. In addition, four samples were collected in the Mauvoisin area (Bagnes valley), in order to compare our results with previous studies and better characterize the regional variability of the RSCM temperatures within the different studied units (Fig. 10c ). Two samples (AP2007 and AP2080) were intentionally collected in close proximity to samples from previous studies (CO0712 from Negro et al. 2013 and #62 from Angiboust et al. 2014, for AP2007; CO092 from Negro et al. 2013 for AP2080) . The comparison between the RSCM temperature obtained for these samples show that RSCM temperature values are comparable between our study and these previous studies (values overlap within a 2σ error range for both locations). Examination of the entire set of RSCM temperatures then available for the samples located in the Série Rousse and the Série Grise shows that no significant difference in temperature exists between these two series. Indeed, in the Hérens valley, these temperatures range from 408 ± 27 C to 467 ± 23 C for the Série Rousse (mean val. = 430 ± 20 C; n = 10) and from 411 ± 17 C to 466 ± 29 C for the Série Grise (mean val. = 441 ± 16 C; n = 8); in the Bagnes valley, between 457 ± 14 C and 494 ± 7 C for the Série Rousse (mean val. = 477 ± 15 C; n = 7) and between 461 ± 4 C and 487 ± 23 C for the Série Grise (mean val. = 475 ± 9 C; n = 8); the few values available in the Anniviers valley (incl. the Moiry valley) range between 441 ± 12 C and 451 ± 5 C for the Série Rousse (mean val. = 447 ± 5 C; n = 3) and between 437 ± 14 C and 458 ± 16 C for the Série Grise (mean val. = 446 ± 10 C; n = 5); see Additional file 2 for details. These data show, however, a significant variation in RSCM temperatures between the different sectors of the study area. The amplitude of this variation is equivalent for the Série Rousse and the Série Grise. This regional variation of the peak temperatures can be explained by differences in tectonic positions (Fig. 14a, b ). Indeed, the most external sectors, characterized by a shallower burial depth, such as those of the Hérens and Anniviers valleys, show lower temperatures than the more internal ones, such as the Bagnes valley, which are characterized by higher burial depths. The dense data set obtained for the Hérens valley (Additional file 3) allows to quantify the distribution and variability of the RSCM temperatures through the whole Schistes Lustrés complex in this sector (Fig. 10a, b ). The samples providing the highest RSCM temperatures are those from the immediate vicinity of the contact between the Série Rousse and the Série Grise. The maximum RSCM temperatures are identical for both series (467 ± 23 C for the Série Rousse and 466 ± 29 C for the Série Grise) and are obtained from the samples located closest to this contact (< 10 m). Figure 10d displays the distribution of RSCM temperatures as a function of their distance from the contact between the Série Rousse and the Série Grise. An increase in temperatures centered on this contact can be clearly evidenced. The relative influence of factors that control the processes of crystallization and recrystallization of organic matter during metamorphism and rock deformation is still not fully established and quantified (e.g. Nakamura et al. 2017; Beyssac et al. 2019 ). Nevertheless, the following causes can be suspected to be responsible for the particular distribution of RSCM temperature evidenced through the contact between the Série Rousse and the Série Grise. (1) A local temperature increase along an Alpine thrust. ( 2 ) Recrystallization of the organic material facilitated by fluid flow in the contact zone; and/or (3) by more intense deformation in the contact zone. These different hypotheses are not mutually exclusive and are all compatible with a contact of tectonic nature, as suggested by previous studies (cf. chap. 2.2) and the observations of the previous chapter (5.2). Regarding the first hypothesis, a temperature increase associated to faulting, and shearing along ductile shear zones, has been documented in numerous natural examples, at various scales and magnitudes of temperature variations (e.g. Nicolas et al. 1977; Scholz 1980; England and Molnar 1993; Leloup and Kienast 1993; Nabelek Hérens valley plotted on a geological cross-section constructed through the area (modified after Escher 1988; Escher et al. 1988 Escher et al. , 1993 Escher et al. , 1994)) ; location indicated in Fig. a . c RSCM temperatures [C] at regional scale: literature data (squares, pentagons and triangles) and data from this study (circles); background map from Fig. 1b (SE zone modified after Steck et al. 1999 Steck et al. , 2015;; Dal Piaz et al. 2015) Argand (1911) , Witzig (1948) , Hagen (1951) , Gouffon and Burri (1997) , Burri et al. (1999) ; locations on Fig. 7a; A . 2001; Stipp et al. 2002; Petroccia et al. 2022) . The mechanism of shear heating (or strain heating) and their resulting thermal anomalies, associated both with brittle and ductile deformation, have been extensively studied through thermomechanical modeling (e.g. Molnar and England 1990; Leloup et al. 1999; Nabelek et al. 2001; Burg and Gerya 2005; Duprat-Oualid et al. 2015; Schmalholz and Duretz 2015; Aharonov and Scholz 2018; Mako and Caddick 2018; Kiss et al. 2019) . The thermal anomaly pattern evidenced by our study across the thrust surface and adjacent areas is compatible, in term of amplitude and spatial extension, with the results of the recent thermomechanical modeling studies from Mako and Caddick (2018) and Kiss et al. (2019) , for example. The respective influences of strain and fluid flow on graphite (re)crystallization, as evoked in the second and third hypotheses, are poorly constrained for natural settings and geological time scales. Both parameters seem, however, to influence and enhance graphitization kinetics as shown by laboratory experiments (e.g. Ross and Bustin 1990; Bustin et al. 1995; Beyssac et al. 2003a; Nakamura et al. 2017 ) and some natural examples (e.g. Luque et al. 1998; Luque and Rodas 1999; Kirilova et al. 2018; Wang et al. 2019; Petroccia et al. 2022) . Regardless of the mutual influence of the factor(s) controlling the evidenced distribution of the RSCM temperatures measured across the Hérens valley, these results emphasize the importance of the tectonic contact of the Série Grise on the Série Rousse and reinforce the observations and conclusions of chap. 5.2. The Schistes Lustrés samples from the Hérens valley with the lowest RSCM temperatures are from metasediments associated with the large ophiolitic masses of the Tracuit -Aiguilles Rouges zone (Fig. 10a, c ; Decrausaz et al. 2021, fig. 2 ). The RSCM temperatures of these samples range from 354 ± 13 C to 402 ± 14 C, they are therefore significantly lower than those of both Série Grise and Série Rousse in the same area, which together range there from 408 ± 27 C to 467 ± 23 C. This contrast in RSCM temperature is of the same order as that described by Manzotti et al. (2021) between the Cornet Unit (373 ± 13 C -403 ± 9 C) and the By Unit (462 ± 12 C -515 ± 23 C) in the Ollomont valley (IT). The Cornet Unit is located in the direct continuation of the Tracuit -Aiguilles Rouges zone, in an identical tectonic position (Fig. 1b ). These two units are therefore probably equivalent. The Low-T slice (360-410 C) of the Tsaté complex highlighted by Angiboust et al. (2014) in this same region as our study (without being mapped) also likely corresponds to these units.
Discussion
Evolène Series -Série Rousse succession compared to stratigraphic series from the Prealps and Western Alps The detailed observations along the basal contact of the Série Rousse presented in chapter 5.1 clearly suggest the stratigraphic nature of this contact, as pointed out in particular by the local identification of a basal conglomerate and a basal hardground, and by the absence of any hint of particular shearing across various sections of the contact. The interpretation of this contact as stratigraphic, as already proposed by e.g. Escher (1988; cf. Fig. 2) , clarifies various points concerning the evolution of the Prepiemont domain (cf. Lemoine 1961; Pantet et al. 2020) , which are discussed below. In particular, the study of the facies distribution within the Série Rousse and the underlying formations of the Mont Fort basement and Evolène Series provides interesting insights regarding the Cretaceous paleogeographic and sedimentological evolution of the Prepiemont domain and the tectonic correlations between the Prepiemont units outcropping in the Pennine Alps and those transported in the Prealps.
Characteristic successions of siliceous limestones and black detrital limestones Different local stratigraphic columns showing the lithological successions outcropping on both sides of the basal contact of the Série Rousse are presented in Fig. 11b for the Mauvoisin area (Bagnes valley); and in Fig. 12b for (Moix and Stampfli 1980, 1981; Schneider 1982; Escher 1988; Allimann 1990; Kramar 1997; Steck et al. 1999; Sartori and Epard 2011; Glassey 2013; Marthaler et al. 2020a the Hérens valley area. A well-developed conglomerate or hardground is observed at the base of the Série Rousse when the underlying Evolène Series is devoid of Mauvoisin marbles (e.g. Fig. 11b : Sa and Ro logs; Fig. 12b : MB, RV and SN logs). This suggests a marked sedimentation gap, that should span from the Late Jurassic to the Late Cretaceous. On the other hand, when the Série Rousse rests on the Mauvoisin marbles, its basal part generally includes dark levels, rich in phyllosilicates and organic matter (e.g. , 6c, 6e ). These successions, characterized by the superposition of light-colored siliceous calcitic marbles, with quartzitic mm-cm thick levels, attributed to the Mauvoisin marbles (cf. chap. 4.2), surmounted by dark calcschists or detrital calcitic marbles of the Série Rousse (cf. chap. 4.1.1), recall the stratigraphic superposition of the Bonave Fm. (Upper Tithonian -Valanginian/ Barremian) and the glauconite-bearing Joux Verte Fm. (Upper Barremian -Middle Turonian) described in the Breccia nappe of the Chablais Prealps (Dall' Agnolo 1997, 2000; Fig. 13a ). In the Briançonnais domain s.l. (incl. Prepiemont), the stratigraphic succession of light-colored, massive limestones (or breccias with light-colored limestone matrix of the same age), of cherty light-colored pelagic limestones, and of darker phyllitic and sometimes anoxic limestones, is in fact very characteristic of the superposition of Upper Jurassic, Lower Cretaceous and "mid"-Cretaceous levels (Bourbon et al. 1979 ). Such successions can be observed in many and less metamorphosed and deformed stratigraphic successions of the Western and Central Alps, e.g. in the Plastic Median Prealps (e.g. Plancherel et al. 2020) , the Roche des Clots Series (e.g. Lemoine et al. 1978) , or the Chabrière Series (Lemoine and Tricart 1986) . The characteristic superposition of these facies on different sections of the contact between the Evolène Series and the Série Rousse constitutes therefore another strong argument to consider this contact as stratigraphic.
Stratigraphic comparison with the Breccia nappe in the Prealps The stratigraphy of the Breccia nappe in the Prealps (Fig. 13a ; e.g. Lugeon 1896; Chessex 1959; Weidmann 1972; Dall' Agnolo 1997 , 2000; Plancherel et al. 1998) shows very strong similarities with the entire Evolène Series. These similarities have been pointed out several times in the literature (Joukowsky 1907; Escher 1988; Kramar 1997; Sartori et al. 2006; Marthaler et al. 2008b; Glassey 2013; Marthaler et al. 2020b ). It has recently been discussed in detail by Pantet et al. (2020) , who highlight a strong correlation between these two stratigraphic successions. The correlations proposed here between the Bonave Fm. and the Mauvoisin marbles (upper part of the Evolène Series), and between the Joux Verte Fm. and the lower part of the Série Rousse, further strengthen the correlation between the Breccia nappe and the whole Mesozoic series associated to the Mont Fort nappe (Fig. 13 ). The few differences between these two stratigraphic successions are minor. The absence of Lower and Middle Triassic levels in the Prealps is simply explained by the detachment of the stratigraphic successions above the Carnian evaporites (Caron in Debelmas 1970; Weidmann 1972) . Differences also appear concerning the thickness of the Jurassic and Cretaceous levels between the two stratigraphic columns of Fig. 13 . They are however not really significant, since these synthetic columns A. Pantet et al. only indicate maximum thicknesses, whereas considerable thickness variations are observable within the series themselves for these levels (Dall' Agnolo 1997 , 2000; Figs. 7, 11, 12) . Regarding the Breccia nappe, these thickness variations are mainly of stratigraphic origin, whereas for the units associated with the Mont Fort nappe, their origin is not only stratigraphic, but also partly tectonic, given the strong Alpine deformation affecting these units. Finally, another difference between the two columns concern the flysch and the melange (Suprabrekzienmelange/ Mélange Supra-Brèche; Dall ' Agnolo 1997 ' Agnolo , 2000;; Mattes Melange; Plancherel et al. 2020) overlying the stratigraphic series of the Breccia nappe (Dall' Agnolo 1997 , 2000) , and for which, no equivalent has been recognized at the top of the Série Rousse. However, the relationship of this melange to the Breccia nappe is possibly tectonic (De Lepinay 1981; Dall' Agnolo 1997) and its potential equivalent at the top of the Série Rousse (as well as the overlying levels) may have been tectonically replaned by the overthrusting of the Upper Penninic units.
Late Cretaceous Strong stratigraphic similarities can therefore be evidenced between the Breccia nappe in the Chablais Prealps and the ensemble formed by the Evolène Series and the Série Rousse. They lead us to consider this ensemble as a single entity, whose stratigraphic successions extend from the basal Triassic to the Upper Cretaceous at least. the Cretaceous with a smaller stratigraphic gap than in adjacent areas. The different points discussed above show strong evidences for the onlap and sealing by the Série Rousse, during the Late Cretaceous, of a paleotopography characterized by fault scarps resulting of the Jurassic development and activity of large synsedimentary extensional faults. The Série Rousse therefore represents the sedimentary continuation of the Evolène Series and should consequently not be considered as an independent allochthonous tectonic unit.
Tectonic limits and units in the Schistes Lustrés complex of the Combin zone Taken together, our observations thus indicate that the Série Rousse constitutes the youngest part of the autochthonous sedimentary cover of the Mont Fort nappe, rather than a basal slice of the Tsaté nappe. They also show that the upper contact of the Série Rousse with the ophiolite-bearing Schistes Lustrés of the Série Grise is clearly of tectonic nature (cf. chaps. 2.2, 5.2, 6) . This contact corresponds to the Alpine thrust separating the Tsaté and Mont Fort nappes. According to our interpretation, the contact between these two nappes is therefore not located at the base of the Série Rousse, but at its top. This contact, which corresponds to the major tectonic limit that separates the Upper Penninic ocean-derived units from the continental margin-derived Middle Penninic units, is thus located within the Schistes Lustrés complex of the Combin zone (Figs. 2, 14) . According to the proposed interpretation, the ensemble constituted by the Evolène Series and the Série Rousse thus forms the relative autochthonous sedimentary cover of the Mont Fort nappe, whereas the Tsaté nappe is restricted to the Série Grise and the Tracuit -Aiguilles rouges zone / Cornet Unit. The thin Frilihorn slices are located at the contact of the two nappes rather than within the Tsaté nappe. This interpretation corresponds in fact to the first hypothesis adopted when these different units were defined (Fig. 2 ; Marthaler and Escher in Masson et al. 1980; Marthaler 1981 Marthaler , 1984;; Escher and Masson 1984; Sartori 1987; Escher 1988; Escher et al. 1988 ) and before the subsequent reinterpretation of this tectonic scheme by some of these authors (e.g. Escher et al. 1993; Sartori and Marthaler 1994; cf. chap. 2.2) . The interpretation of the tectonic position of the Frilihorn slices, at the base rather than within the Tsaté nappe, is interesting with respect to the questions of the origin and tectonic attribution of these slices. In the studied area, north of the Dent Blanche klippe, all the observed Frilihorn slices are located along the contact between the
The basal unconformity: progressive infill and sealing of the remnants of the Jurassic fault scarps The basal contact of the Série Rousse is characterized by an unconformity described in chap. 5.1.1. This unconformity has led various authors (Escher et al. 1993; Sartori and Marthaler 1994) to consider this contact as being of a tectonic nature. All our observations however consistently show that the contact is primarily stratigraphic (cf. chaps. 5.1. 2-5.1.4, 7.1) and that the unconformity can be explained by deposition on a paleorelief of former syn-sedimentary faults (Figs. 11c, 12c) , following a model already developed by Pantet et al. (2020) . These syn-sedimentary faults, mostly active during the Jurassic, delimit small basins formed by tilted blocks (halfgrabens), whose movement is at the origin of the locally large breccia accumulations observed in the Evolène Series. The thinness of the breccia deposits in the Série Rousse (in general < 10 cm, never exceeding 3 m) seems to indicate that these faults were no longer active or less active during the Late Cretaceous. A submarine relief formed by the Jurassic fault scarps should have remained, allowing the local erosion of the Evolène Series and of various Paleozoic levels of the Mont Fort basement. The activity of Cretaceous faults or rejuvenation of Jurassic faults during the Cretaceous has nevertheless been documented in other areas of the Alps (e.g. Claudel et al. 1997; Michard and Martinotti 2002; Bertok et al. 2012; Cardello and Mancktelow 2014; Michard et al. 2022) . Our detailed mapping (Figs. 7a, 12a) shows that the different lithological successions along the basal contact of the Série Rousse (e.g. Figs. 11b, 12b) , as well as their distribution, are compatible with the hypothesis of a progressive filling of such half grabens by the Série Rousse. For example, the conglomerate, locally present at the base of the Série Rousse, is better developed where the Evolène Series is absent or reduced to a small thickness. Following our restoration attempts of the structures preceding the Alpine collision (Figs. 11c, 12c) , the areas yielding these basal conglomerates would correspond either to areas characterized by the sedimentation of the Série Rousse directly on ancient fault scarps (e.g. Fig. 11b , PV, Sa and Ro logs; Fig. 12b , MP log), or to the upper parts of tilted blocks (e.g. Fig. 12b , MB and SN logs). The area where a hardground is observed and document a large stratigraphic gap (Triassic levels and Mauvoisin marbles missing; Fig. 12b , SN log), would correspond, following the restoration attempt of Fig. 12c , to the highest part of a tilted-block. Areas where the Série Rousse overlies the Mauvoisin marbles and shows dark facies (e.g. Fig. 11b, BP log; Fig. 12b, Vi, PT and SC logs) correspond, in contrast, to the depocenters of the half graben basins, where sedimentation continued during Série Rousse and the ophiolite-bearing Schistes Lustrés (which is locally redoubled by isoclinal folds; Fig. 9c ). These observations actually seem hardly compatible with the hypothesis of an Adriatic origin of these slices (e.g. Staub 1942d; Caby et al. 1978; Dal Piaz 1999; Froitzheim et al. 2006; Pleuger et al. 2007; Passeri et al. 2018) . It is indeed extremely difficult to imagine a mechanism able to insert slices of Adriatic origin in this position without inserting ophiolite-bearing Schistes Lustrés below these slices. However, a provenance from the Briançonnais s.str. swell and a southward emplacement, as proposed by Scheiber et al. (2013) , also seems unlikely to be compatible with the widespread breccia layers observed within the calcitic marbles of these slices (cf. chaps. 2.3.2, 5.2); and with the top-NW shear sense associated to the main schistosity that is observed in some localities (Fig. 9e ). According to the observations above, a Prepiemont origin (i.e. derived from the southern distal margin of the Briançonnais s.l. domain; Fig. 15b ), as proposed for example by Elter (1960 Elter ( , 1972;; Dal Piaz (1974) , Bearth (1976) , Marthaler (1984) , Escher (1988) , seems, therefore, more likely. Considering such a Prepiemont origin, two different possibilities concerning the origin and tectonic attribution of the Frilihorn slices could be envisaged: (1) a more distal origin than the Mont Fort nappe on the distal Prepiemont margin, the Frilihorn slices would hence constitute an independent tectonic unit, thrusted along the base of the Tsaté nappe; (2) an origin corresponding to the most internal part of the Mont Fort nappe, folded and boudinaged under the Tsaté basal thrust (similarly e.g. to the Helvetic Drône anticline under the Penninic thrust; Steck et al. 1989; Escher et al. 1997; Cardello et al. 2019) . The existence of an Alpine tectonic contact within the ophiolite-bearing Schistes Lustrés (i.e. the Tsaté nappe as redefined above) has been postulated by Angiboust et al. (2014) . Its existence has been recently confirmed by Manzotti et al. (2021) based on metamorphic arguments. Indeed, on each sides of this contact in the Ollomont valley (IT), both the RSCM temperatures and the metamorphic paragenesis are strongly contrasted. In this sector, the lower part of the Schistes Lustrés complex (By Unit) shows parageneses characterized by the presence of pseudomorphs after lawsonite, aragonite inclusions in titanites and the local presence of small garnets in quartzitic schists. Its peak P-T conditions are estimated at ca. 16-17 kbar and 460-480 C. In the Cornet Unit, which represents there the uppermost unit of the Schistes Lustrés complex, the parageneses mentioned above are completely absent and the peak P-T conditions are estimated at ca. 8 ± 1 kbar and 370-400 C. According to these authors, the metamorphic evolution is similar between the Cornet Unit and the overlying Dent Blanche nappe. The upper limit of the Schistes Lustrés complex of the Combin zone would therefore not correspond to an important metamorphic limit. The same seems to be true for the lower limit of this complex. Indeed, as highlighted above, this limit is stratigraphic in nature and is internal to the Mont Fort nappe, which is not compatible with a metamorphic limit. Available data concerning the metamorphic evolution of the different units composing this nappe are consistent with this interpretation (sodic amphiboles, chloritoid, epidotes and HP white micas in the Paleozoic basement ; Schaer 1960; Bearth 1963; Thélin et al. 1994; Steck et al. 2001; Bousquet et al. 2004 ; Evolène Series RSCMT ranging from 484 ± 31 C to 480 ± 34 C, Table 1 , Fig. 10c ; Série Rousse RSCMT ranging from 408 ± 27 C to 494 ± 7 C, Additional file 2; pseudomorphs after lawsonite in the Série Rousse in the Bagnes valley, Besson 1986 and in the Ollomont valley, P. Manzotti, pers. com.) . The limit between the Mont Fort and Tsaté nappes (i.e. between the Série Rousse ± Frilihorn slices and the Série Grise), although clearly of tectonic nature, neither corresponds to an important metamorphic limit (cf. chap. 6). Contrary to the hypothesis of Angiboust et al. (2014) , the intensity of metamorphism does not appear to increase between the middle and basal parts of the Schistes Lustrés complex. The most important metamorphic boundary in the whole Mont Fort, Tsaté and Dent Blanche nappe stack therefore corresponds to the limit between the Série Grise and the Tracuit -Aiguilles Rouges zone / Cornet Unit. The question of the origin of such a metamorphic discontinuity is beyond the scope of this article and will not be further discussed here. Although the Schistes Lustrés complex of the Combin zone has been considered by many authors as a single tectonic entity, the above data and discussions show that it is instead composed of different tectonic units, separated by (1) a major tectonic contact that corresponds to the Middle-Upper Penninics limit, separating the lower Escher (1988) , Escher et al. (1988 Escher et al. ( , 1993 Escher et al. ( , 1994)) , Schaer (1960) and middle units of this complex, i.e. the Série Rousse and the Série Grise respectively, and ( 2 ) by a major metamorphic discontinuity, separating the middle from the upper unit of this complex (Tracuit -Aig. Rouges zone / Cornet Unit). It is particularly interesting to note that these two contacts do not correspond to marked lithological limits, they are even sometimes extremely inconspicuous, which may explain why their importance has been underestimated by most authors. In the light of these new data, the structure of the Combin zone is thus comparable to that of the Schistes Lustrés of the Western Alps, also characterized by a tectonic juxtaposition of different units showing distinct metamorphic evolutions (e.g. Fudral et al. 1987; Rolland et al. 2000; Plunder et al. 2012; Agard 2021; Herviou et al. 2022) .
M o n t F o r t n a p p e S i v i e z -M i s c h a b e l n a p p e
SC
Cretaceous-Paleogene sedimentation along the Briançonnais-Prepiemont margin and Piemont basin The Briançonnais s.str. domain, its hyperextended SEmargin (Prepiemont domain; Lemoine 1961 ) and the Piemont basin are characterized by a deep-water, clastic, hemipelagic and pelagic sedimentation throughout the Cretaceous (Fig. 15 ). Thus, until the end of this period, these realms remained essentially unaffected by the subduction and the onset of the Alpine collision that started along the Adriatic margin and southern Piemont-Liguria basin at that time. The onset of the convergence is attested since the late Cenomanian to early Turonian by the first Alpine flysch units, containing material of the Adriatic margin and now constituting the Simme nappe (Caron et al. 1989; Gasinski et al. 1997) , and by the age of the prograde HP metamorphism starting at ca. 85 Ma in the Adriatic and Piemont derived units of the Pennine Alps (Skora et al. 2009; Rubatto et al. 2011; Manzotti et al. 2014; Regis et al. 2014) . Stratigraphic data from the Briançonnais s.l. series in the Swiss Prealps (Fig. 15a ) show that pelagic to hemipelagic sedimentation extends into the Paleocene in the external part of the Prepiemont domain (Couches Rouges of the Breccia nappe; Dousse 1965; Dall' Agnolo 1997) and into the early Eocene in the Briançonnais s.str. domain (Chenaux Rouges Fm.; Guillaume 1986) . It is then relayed by flysch deposition, until middle Eocene (Guillaume 1986; Dall' Agnolo 1997; Hable 1997) . Alpine metamorphism and deformation in the Briançonnais s.l. and surrounding units of the Pennine Alps are dated to the late Eocene, with ages ranging in Western Switzerland between 37-40 Ma for the Prepiemont domain and 35-40 Ma for the Briançonnais s.str. domain (e.g. Markley et al. 1998; Gebauer 1999; Angiboust et al. 2014) . The sedimentation gaps, sometimes large, revealed at different levels in the Cretaceous and Paleocene of these series (Fig. 15a ) are interpreted as resulting from the progression of the lithospheric bulge associated with the progressive subduction of the Piemont domain under the Adriatic plate (Stampfli et al. 1998) . The sedimentary evolution during the Late Cretaceous -Paleogene is less constrained in the internal Prepiemont domain and Piemont domain (corresponding to the domains of deposition of the Série Rousse and Série Grise respectively), given the poor preservation of microfossils in the Pennine Alps and the lack of complete corresponding series in the Prealps. The few biochronological data available indicate a Cenomanian to earliest Maastrichtian minimal age for the Série Rousse (cf. chaps. 2.3.3, 4.1.2) and a Cenomanian to Santonian minimal age for the Série Grise (cf. chap. 2.3.1). The presence of well-developed dark anoxic levels in these series may, in turn, argue for their partly "mid"-Cretaceous age. The ages that can be inferred for these series are thus comparable with those indicated by Dall' Agnolo (1997 , 2000; cf. Fig. 15a ) for the Joux Verte Fm. (late Barremian -middle Turonian) and for the base of the Couches Rouges from the Breccia nappe (Campanian -Paleocene) as well as for the Couches Rouges included in the Mattes Melange (or Supra-Breccia Melange; late Campanian -early Paleocene; Dall ' Agnolo 1997 ' Agnolo , 2000;; Plancherel et al. 2020) . As the Mattes Melange contains lenses of Couches Rouges and lenses of Helminthoid Flysch and is intercalated between the Breccia nappe and the upper Prealps nappes (Badoux 1962; Caron 1966 Caron , 1972;; Caron and Weidmann 1967; Dall' Agnolo 1997) , the origin of these lenses must therefore correspond either to the internal part of the basin of the future Breccia nappe or to a more internal domain, i.e. to the inner part of the Prepiemont domain or the Piemont domain. Lithological successions unambiguously corresponding to Couches Rouges or flysch units have however not been recognized at the top of the Série Rousse or Série Grise. The lithologies observed in these series differ from the Couches Rouges of the Médianes Prealps and of their metamorphic equivalents of the Barrhorn Series, by systematically higher quartz and organic matter contents (in the Couches Rouges and their metamorphic equivalents, quartz content is usually < 2-5% and CM is very scarce; Guillaume 1986; Sartori 1990; Dall' Agnolo 1997) . The Couches Rouges described in the Breccia nappe and in the Mattes Melange seem however to have a higher detrital input (Dousse 1965; Dall' Agnolo 1997) and could partly correspond to the upper part of the Série Rousse (Figs. 13 and 15 ). The lithologies of the upper part of the Série Rousse are also further distinguished from the Breccia Flysch, by the scarcity of graded beds and the non-cyclic character of the sedimentation. It is nevertheless possible that the Couches Rouges and flysch, included as lenses in the Mattes Melange, may have first been deposited at the top of the Série Rousse and/or of the Série Grise, but that these sequences were then locally detached and transported to the Prealps. Such a detachment seems to have affected the Breccia nappe in the Chablais Prealps, where it would have occurred at the level of the of the "mid"-Cretaceous anoxic shales (De Lepinay 1981; Dall' Agnolo 1997) . It is reflected by the absence of levels younger than the middle Turonian and by an important unconformity at the top of the internal part of the Breccia nappe. A general slope from the Briançonnais swell towards the Piemont basin (Fig. 15b ) is documented by an overall proximal to distal and upward fining of the Cretaceous detrital sediment component. Indeed, during the whole period from the Early Cretaceous to the early Late Cretaceous, the detrital input is significantly larger in the Prepiemont domain than in the Briançonnais s. str. domain (cf. Dall' Agnolo 1997 , 2000; Plancherel et al. 2020) . While submarine topographic highs of the Briançonnais were sheltered from such a detrital input and were the site of hemipelagic to pelagic sedimentation (which continued until the end of the Couches Rouges deposition), sedimentation in the Prepiemont domain was characterized by an input of quite mature quartz and detrital carbonate sands and episodic breccias intercalations. The presence of large fault scarps in the Prepiemont domain, preserved from the Jurassic extension and exposing both lower Mesozoic and Paleozoic rocks at the seafloor (cf. chap. 7.2), are very likely to constitute the main source of this detrital input in the Prepiemont Cretaceous formations.
Conclusion Our study shows that the Schistes Lustrés complex of the Combin zone in Western Switzerland consists of several lithologically distinct and tectonically independent series. The lower Schistes Lustrés unit, the Série Rousse, is non-ophiolitic and consists mainly of detrital calcitic marbles and calcschists with continental affinities. We argue that the discontinuity characterizing the basal contact of these series results rather from the passive onlap and progressive sealing of remnants of Jurassic extensional fault scarps, than from a tectonic contact between different slices emplaced in an accretionary prism context, as previously proposed. The stratigraphic nature of the basal contact of the Série Rousse is attested by (1) the occurrence in several sectors of a basal conglomerate, sometimes reworking directly underlying formations of the Mont Fort nappe, (2) the local preservation of a hardground at the actual base of the series, and (3) the absence of particular shearing along different sections of the contact. The Série Rousse therefore belongs to the relative autochthonous Mesozoic cover of the Mont Fort nappe, which it forms together with the Evolène Series (cf. Pantet et al. 2020) , as previously proposed by Escher (1988) for example. The stratigraphic sequence composed by the superposition of the Evolène Series and the Série Rousse shows very strong similarities with the stratigraphy of the Breccia nappe in the Prealps. The comparison of the Série Rousse with the Breccia nappe, which is better constrained biochronologically, as well as the discovery in the Série Rousse of a form corresponding to Globotruncana neotricarinata, allows us to better constrain its age. It probably extends from the Early Cretaceous (Aptian?) to the Late Cretaceous (Campanian to earliest Maastrichtian?). The median Schistes Lustrés unit, the ophiolite-bearing Série Grise, overlies the Série Rousse by a tectonic contact corresponding to the Upper/Middle Penninic limit. It is underlined by tectonized slices (Frilihorn Series), whose origin corresponds either to the internal part of the Mont Fort nappe, or to a more internal Prepiemont unit. The performed RSCM analyses show that the recrystallization of the organic matter progressively increases towards this contact. RSCM temperatures calculated in the Hérens valley for samples taken in the immediate vicinity of the contact (< 10 m) reach 467 ± 23 C for the Série Rousse and 466 ± 29 C for the Série Grise, whereas the lowest values obtained for both series in this same valley come from the areas most distant from the contact (408 ± 27 C and 411 ± 17 C respectively). This temperature increase centered on the contact may reflect a shear heating effect, associated with the intense strain localized along this major tectonic contact. The upper unit of the Schistes Lustrés complex, the Tracuit -Aiguilles Rouges zone, very likely corresponds to the Cornet Unit individualized by Manzotti et al. (2021) in the Ollomont valley. Our data indicate that the metamorphic limit highlighted by these authors at the base of the Cornet Unit is also found at the base of the Tracuit -Aiguilles Rouges zone. This limit appears as the most important metamorphic discontinuity inside the Mont Fort, Tsaté and Dent Blanche nappe stack. Although the Schistes Lustrés complex of the Combin zone has been regarded by many authors as a single tectonic entity, our study thus confirms and details that it is instead composed of different tectonic units separated by major metamorphic and/or tectonic contacts. Our study moreover clearly documents that the Schistes Lustrés consist of both sediments deposited on oceanic crust, which locally show preserved stratigraphic contacts with ophiolitic or serpentinized sub-continental mantle slivers, as well as of sediments still resting stratigraphically on a former hyper-extended continental margin.
RSCMT Temperature calculated following RSCM method (peak temperature) TL Transmitted light UHP Ultra high pressure [Geographic coordinates] refer to the Swiss grid MN95 Fig. 3 3 Fig. 3 Lithologies of the ophiolite-bearing Schistes Lustrés of the Combin zone. a Characteristic aspect of the Série Grise calcschists; Les Haudères [2′604′860/1′104′050]. b Graded beds of greenish to violaceous volcanosedimentary arkose interbedded in calcschists; Adlerflüe [2′620′290/1′110′560], Turtmann valley. c Decimetric level of greenish calcitic marble outcropping at a few meters from a plurihectometric-long ophiolitic lens (serpentinites, metabasites) of the Série Grise. Detrital mm-sized grains of ophiolitic material are visible under the pencil; Chanrion hut area [2′595′195/1′087′695], Bagnes valley. d Arkosic calcitic marble at the contact of a hectometric-long serpentinite lens. Note the presence of cm-sized serpentinite clasts and the green color of the ophiolitic arkosic detritus; 3 m from Fig. c. e Quartzmicaschists levels (1-2 m thick) interpreted as metaradiolarites (Staub 1942c; Burri et al. 1999) and locally rich in piemontite (sometimes associated to braunite); Chanrion hut area [2′595′165/1′087′700]; Mn-garnets and blue amphiboles are visible in the same level, 200 m to the NNW. f Serpentinite breccia observed at the contact between calcschists of the Série Grise and a plurihectometric serpentinite lens marking the contact with the Série Rousse; Giétro [2′594′360/1′094′290], Bagnes valley
Fig. 4 4 Fig. 4 Lithologies of the Série Rousse. a Typical glossy metallic grey appearance of the schistosity surfaces of the Série Rousse calcschists; Meina pass [2′599′280/1′105′330], Hérens valley. b Alternating grey calcitic marble and russet calcschist levels in the Série Rousse; dolomitic clasts are visible in the same outcrop; Rochers du Bouc [2′595′210/1′099′620], Dix valley. c Centimetric clasts in the Série Rousse, mainly formed by dolomites (pencil), limestones and minor quartzschists (out of the picture); Pic d' Artsinol [2′598′780/1′107′050]. d Microscopic aspect of a quartzitic marble from the base of the Série Rousse. CL: cathodoluminescence; PPL: plane polarized light. Well sorted rounded detrital quartz grains appear in violet in CL; dolomitic grains show mauve to dull orange colors; overgrowth (probably burial) cements are bright orange; zoning and/or dark rims are discordant with respect to the twinning (PPL) and could represent original variations of Mn 2+ incorporation; Pic d' Artsinol [2′599′170/1′108′585]. e Dark levels of calcschists, calcitic marbles and dolomitic breccia (close-up picture) from the Série Rousse; La Tour [2′605′440/1′104′770], Hérens valley. f Rhombohedral automorph mineral observed in various thin sections of dark micritic calcitic marbles of the Série Rousse. Raman spectra indicate feldspar; Pic d' Artsinol [2′598′700/1′108′400]. Similar rhombohedron resulting from the silicification of automorph dolomite crystals are described in dark micrites of the "mid"-Cretaceous Joux Verte Fm. of the Breccia nappe (Dall' Agnolo 1997). g Dark non-carbonated schists outcropping 20 m above the base of the Série Rousse at La Vieille [2′602′800/1′105′540], Hérens valley
Dark layers are interbedded at the base of the Série Rousse in several localities of the study area. They are formed of dark, micaceous and graphitic calcschists, which alternate with dark calcitic marbles and local breccias (Fig.4d). Such layers form the base of the Série Rousse, for example, in the area of Mauvoisin in the Bagnes valley [2′592′810/1′094′670; 2′593′490/1′093′710]; and of the Pic d' Artsinol [2′598′660/1′108′430; 2′599′150/1′108′360]1′104′770] and La Vieille [2′602′680/1′105′660; 2′602′800/1′105′530] in the Hérens valley. In the last locality, dark calcschists, about 20 m thick, are overlain by black siliceous and non-carbonaceous schists (Fig.4f;
Fig. 5 ( 5 Fig. 5 (See legend on previous page.)
Fig. 6 6 Fig. 6 Characteristic facies of the Mauvoisin marbles. a, b Slightly detrital calcitic marble with siliceous mm-cm thick bands; Mauvoisin [2′592′650/1′094′510]. c Similar lithology at La Tour [2′605′310/1′104′860], Hérens valley. d Detrital calcitic marble with mm-cm thick siliceous bands and sparse cm-dm thick limestone clasts (dolomitic clasts appear out of the picture); Giétro [2′593′690/1′092′930], Bagnes valley. e Alternating layers of detrital calcitic marble and dolomitic microbreccia; 10 m from Fig. c. f, g Details of the outcrops of Figs. aand b. In g sharp borders are observed at the contact between the siliceous bands and the surrounding marble, whereas a progressive transition is visible to the more carbonaceous inner parts of these bands. As this characteristic is typical of cherts that are not fully silicified in their inner parts, it supports the biogenic nature of these siliceous bands
2 SérieFig. 7 a 27 Fig. 7 a Detailed geological map of the Mauvoisin area, Bagnes valley; after observations from this study and maps from Baillifard (1998), Burri et al. (1998), Steck et al. (1999) and Dal Piaz et al. (2015). Topographic base Swisstopo 2016; contour intervals: 20 m. 1, 2, 3, 4 refer to Fig. b; Locations of the cross-sections of Fig. 11a are indicated by A, A' , B, B'; Fig.x refer to pictures from other figures; locations of the stratigraphic columns of Fig. 11b are indicated in green. b Types of basal contacts of the Série Rousse in the area covered by the map. c Fold axis and stretching directions in the area covered by the map
Fig. 8 Fig. 8 ( 88 Fig. 8 Conglomerates and hardground from the basal contact of the Série Rousse. a Contact between a polymict conglomerate forming the base of the Série Rousse (russet phyllitic and quartzitic calcitic matrix) and a polymict (Upper Jurassic?) breccia from the Evolène Series. The conglomeratic base of the Série Rousse is only of a few meters thick. A gradual transition is observed with the overlying calcschists of the same series. Rionde de Vendes [2′599′180/1′108′600], Dix valley. b Overturned contact between the Mont Fort Paleozoic basement (Métailler Fm.) and a polymict conglomerate forming the base of the Série Rousse and gradually passing to the typical calcschists of these series (base of the outcrop); Col du Vasevay [2′594′090/1′097′560], Bagnes valley. c Close-up view on locally abundant green clasts of the basal conglomerate; their lithology is identical to that of the overlying Paleozoic metabasites of the Métailler Fm. d Close-up view on clasts of the same conglomerate made of dolomites, limestones and breccias (dolomitic clasts, calcitic matrix). e Basal conglomerate of the Série Rousse showing dm-sized clasts made of the same lithology than the adjacent Tabular Quartzites of the Evolène Series; Col des Roux [2′595′580/1′102′090], Dix valley. f Oxidized crust (hardground) marking the base of the Série Rousse over a light grey (Upper Jurassic?) calcitic marble of the Evolène Series; the dolomitic clast visible at the very base of the Série Rousse shows a rounded shape indicative of the locally low strain and the absence of particular shearing along the contact (the clast is truncated orthogonally to its elongation), as well as the rolling of the clast; Pic d' Artsinol area [2′599′250/1′107′940]. g Basal hardground of the Série Rousse; same location. h Oxidized clasts (reworked hardground) observed in the Série Rousse basal conglomerate; Pic d' Artsinol area [2′600′115/1′105′950]; [2′600′210/1′105′840]; [2′600′100/1′105′965]. i Occurrences of the basal conglomerate and basal hardground of the Série Rousse (map from Fig. 1b) (See figure on next page.)
Fig. 9 9 Fig. 9 Upper contact of the Série Rousse with the ophiolite-bearing Série Grise and the Frilihorn slices. a Tectonized Frilihorn carbonates (partly dolomitic) marking the contact between the Série Rousse and the Série Grise; La Sage [2′606′330/1′105′640], Hérens valley. b Frilihorn dolomitic boudins along the same contact; Plan Tsardon [2′603′400/1′105′470], Hérens valley. c Thin Frilihorn tectonized and folded bands (polyphase folding of a single band); Meina pass area [2′599′360/1′104′680], Hérens valley. d Sharp contact between Frilihorn dolomites and Série Grise metabasites; the Série Rousse is outcropping a few meters below; Palantse de la Cretta west face [2′600′990/1′104′380], Hérens valley. e Strongly sheared Série Rousse calcschists, 3 m below its upper contact with the Série Grise; the outcrop is parallel to the marked stretching lineation; asymmetric folds and C' structures indicate a top to the NW shear-sense; S: schistosity; C': extensional shear bands; Mel de la Niva [2′601′850/1′105′750], Hérens valley
Fig. 10 10 Fig. 10 Geothermometry by Raman spectroscopy of carbonaceous material (RSCM) in the Schistes Lustrés and adjacent lithologies across Hérens valley and surrounding areas of SW Switzerland and NW Aosta valley (IT). a RSCM temperatures [C] across Hérens valley: data from this study (circles) compared with available data from the literature (squares and triangles); background map from Fig. 1b. b RSCM temperatures [C] acrossHérens valley plotted on a geological cross-section constructed through the area (modified after Escher 1988; Escher et al. 1988 Escher et al. , 1993 Escher et al. , 1994)) ; location indicated in Fig.a. c RSCM temperatures [C] at regional scale: literature data (squares, pentagons and triangles) and data from this study (circles); background map from Fig.1b(SE zone modified after Steck et al. 1999 Steck et al. , 2015;; Dal Piaz et al. 2015) . d RSCM temperatures [C] from Figs. a-b (Hérens valley) plotted against the distance of the outcrops to the contact between the Série Rousse (SR) and the Série Grise (SG); distances are calculated orthogonally to the mean local orientations of the structures (cf. Additional file 3); T error bars: 1σ; Dist. error bars: 75%
Fig. 11 a 11 Fig. 10 Geothermometry by Raman spectroscopy of carbonaceous material (RSCM) in the Schistes Lustrés and adjacent lithologies across Hérens valley and surrounding areas of SW Switzerland and NW Aosta valley (IT). a RSCM temperatures [C] across Hérens valley: data from this study (circles) compared with available data from the literature (squares and triangles); background map from Fig. 1b. b RSCM temperatures [C] acrossHérens valley plotted on a geological cross-section constructed through the area (modified after Escher 1988; Escher et al. 1988 Escher et al. , 1993 Escher et al. , 1994)) ; location indicated in Fig.a. c RSCM temperatures [C] at regional scale: literature data (squares, pentagons and triangles) and data from this study (circles); background map from Fig.1b(SE zone modified after Steck et al. 1999 Steck et al. , 2015;; Dal Piaz et al. 2015) . d RSCM temperatures [C] from Figs. a-b (Hérens valley) plotted against the distance of the outcrops to the contact between the Série Rousse (SR) and the Série Grise (SG); distances are calculated orthogonally to the mean local orientations of the structures (cf. Additional file 3); T error bars: 1σ; Dist. error bars: 75% (See figure on next page.)
Fig.11a Geological cross-sections through the Mauvoisin area, Bagnes valley; modified after Argand (1911) , Witzig (1948) , Hagen (1951) , Gouffon and Burri (1997) , Burri et al. (1999) ; locations on Fig.7a; A [2′593′530/1′097 '900]; A' [2′596′320/1′093′260]; B [2′591′460/1′096′700]; B' [2′594′410/1′091′790]; locations of the stratigraphic columns of Fig. b are indicated in green. b Characteristic lithostratigraphic successions through the base of the Série Rousse across the study area; scales differ between the stratigraphic columns (grey strokes represent ca. 20 m); triangles indicate breccia levels; circles, conglomerates; black points, detrital quartz; black dashes, dark calcschists; black lines, black schists. Stratigraphic columns acronyms and coordinates: PV [2′594′390/1′097′950]-[2′594′400/1′097′970], tectonically overturned; Sa [2′594′555/1′097′430]-[2′594′580/1′097′430]; Ro [2′594′110/1′097′570]-[2′594′090/1′097′560], tectonically overturned, pictures Fig. 8b-d; GR [2′593′780/1′097′055]-[2′593′780/1′097′045], tectonically overturned; DG [2′593′050/1′094′945]-[2′593′065/1′094′930], tectonically overturned; BP [2′593′130/1′094′830]-[2′593′160/1′094′860]; EG [2′593′710/1′092′930]-[2′593′615/1′092′930], tectonically overturned, Fig. 6d from the lower part of the profile. c Proposed pre-orogenic restoration (Late Cretaceous) of the cross-section of a; approximate pre-orogenic locations of the lithostratigraphic successions of Fig. b are indicated in green (See figure on next page.)
Fig. 11 (Fig. 12 a 1112 Fig. 11 (See legend on previous page.)
Fig.12a Bedrock map of the Evolène area (Hérens valley); compiled after data from this study and existing maps(Moix and Stampfli 1980, 1981; Schneider 1982; Escher 1988; Allimann 1990; Kramar 1997; Steck et al. 1999; Sartori and Epard 2011; Glassey 2013; Marthaler et al. 2020a; Swisstopo Geocover); background altitude model Swisstopo; locations of the stratigraphic columns of Fig. b are indicated in green. b Characteristic lithostratigraphic successions through the base of the Série Rousse along the Hérens valley; scales differ between the stratigraphic columns (grey strokes represent ca. 20 m); triangles indicate breccia levels; circles, conglomerates; black points, detrital quartz; black dashes, dark calcschists; black lines, black schists; brown line, hardground. Stratigraphic columns acronyms and coordinates: Co [2′600′850/1′108′710], tectonically overturned, cf. Allimann (1990); Be [2′605′485/1′108′880]-[2′605′510/1′108′870], cf. Glassey (2013); MB [2′605′260/1′107′190]-[2′605′320/1′107′130]; RV [2′605′130/1′106′900]-[2′605′180/1′106′880]; Ts [2′600′050/1′107′940]-[2′599′790/1′107′790]; Vi [2′602′710/1′105′660]-[2′602′800/1′105′520], tectonically overturned, Fig. 4g from the center of the profile; PT [2′602′950/1′105′540]-[2′603′415/1′105′460], Fig. 9b from the top of the profile; SC [2′605′545/1′105′230]-[2′605′740/1′105′185]; SN [2′599′285/1′107′975]-[2′599′290/1′107′895], Fig. 8f-g from the center of the profile; MP [2′599′250/1′105′470]-[2′599′235/1′105′450]. c Pre-orogenic restoration attempt, reproducing lithological contacts and successions observed and reported in Fig. a-b (See figure on next page.)
Fig. 11b: DG, BP and EG logs; Fig. 12b: Ts, Vi, PT and SC logs). Good examples of such successions can be observed near the Mauvoisin Hotel at [2′592′710/1′094′620] and [2′592′820/2′094′650]; and in the Hérens valley, in the vicinity of La Vieille (Fig. 12, Vi log; Marthaler et al. 2020a, 2020b), near the St-Christophe chapel (Fig. 12, SC log) and SE of La Tour between [2′605′290/1′104′860] and [2′605′460/1′104′760] (Figs. 4e
Fig. 12 (Fig. 13 a 1213 Fig.12(See legend on previous page.)
Fig. 14 14 Fig. 14 Reinterpreted schematic cross-sections and tectonic map of the studied area; Ar: Ardon nappe; DB B : Dent Blanche basement; Fr: Frilihorn slices; Mo: Morcles nappe; R: Ruitor basement; SC: Sion-Courmayeur zone; SM B : Siviez-Mischabel basement; SM M : Siviez-Mischabel Mesozoic; UH: Ultrahelvetic nappes; Wi: Wildhorn nappe; ZH: Zone Houillère; ZH B : Zone Houillère basement; ZH M : Zone Houillère Mesozoic. a Schematic geological cross-section through the Hérens valley; modified after Escher (1988) , Escher et al. (1988 Escher et al. ( , 1993 Escher et al. ( , 1994)) , Schaer (1960) ; location indicated in Fig. c. b Schematic geological cross-section through the Bagnes valley; location indicated in Fig. c. c Tectonic map of the upper Bagnes, Dix, Hérens and Anniviers valleys (SW Switzerland); compiled after data from this study and existing maps (see Fig. 1b for references)
Fig. 14 Reinterpreted schematic cross-sections and tectonic map of the studied area; Ar: Ardon nappe; DB B : Dent Blanche basement; Fr: Frilihorn slices; Mo: Morcles nappe; R: Ruitor basement; SC: Sion-Courmayeur zone; SM B : Siviez-Mischabel basement; SM M : Siviez-Mischabel Mesozoic; UH: Ultrahelvetic nappes; Wi: Wildhorn nappe; ZH: Zone Houillère; ZH B : Zone Houillère basement; ZH M : Zone Houillère Mesozoic. a Schematic geological cross-section through the Hérens valley; modified after Escher (1988) , Escher et al. (1988 Escher et al. ( , 1993 Escher et al. ( , 1994)) , Schaer (1960) ; location indicated in Fig. c. b Schematic geological cross-section through the Bagnes valley; location indicated in Fig. c. c Tectonic map of the upper Bagnes, Dix, Hérens and Anniviers valleys (SW Switzerland); compiled after data from this study and existing maps (see Fig. 1b for references) (See figure on next page.)
Fig. 14 ( 14 Fig. 14 (See legend on previous page.)
Fig. 15 a 15 Fig. 15 a Correlations between the lithostratigraphic formations of the Breccia, Médianes, Mont Fort and Tsaté nappes (Prealps and Pennine Alps) for the Jurassic, Cretaceous and Paleogene periods; in this representation, only the time-extensions of the formations are indicated, independently of their actual thickness; modified after Dall' Agnolo (1997, 2000); Mosar et al. (1996, fig. 4); Stampfli et al. (1998, 2002); Plancherel et al. (1998, 2020); Decrausaz et al. (2021); Marthaler et al. (2020b); brown lines represent hardgrounds; blue dashed lines indicate probable tectonic contacts; Ccsl: Calcaires compacts et sublithographiques; MB: Mytilus Beds; He: Heiti Fm. b Restoration attempt of the South Briançonnais -Prepiemont margin at Late Cretaceous Epoch, before the onset of Alpine deformation in that area; modified after Stampfli and Marthaler (1990); Stampfli et al. (1998, 2002); Mohn et al. (2010); Pantet et al. (2020); UB: Ultrabriançonnais domain
). In the Schistes Lustrés of the Western Alps, similar quartzschist levels have yielded radiolarians of Callovian to Middle Kimmeridgian age (De Wever and Caby 1981; Deville 1987; De Wever et al. 1987 ). 2.3.1.5 Meta(ultra-)basites Among the ophiolitic inter- calations in the Schistes Lustrés of the Combin zone, metabasites are the most abundant. Their chemistry is generally of MORB type (Dal Piaz et al. 1981; Beccaluva et al. 1984; Decrausaz et al. 2021) and their parageneses (epidote + albite + chlorite + titanite ± actinolite/tremo-lite ± phengite ± apatite ± calcite; e.g. Angiboust et al. 2014; Marthaler et al. 2020b; Decrausaz et al. 2021) are typical of the greenschist facies. An early, largely ret- romorphosed, blueschist facies paragenesis is locally preserved (e.g. Dal Piaz and Ernst 1978; Ayrton et al. 1982; Sperlich 1988; Martin and Cortiana 2001; Angi- boust et al. 2014). The metabasalts (prasinites, ovardites) form levels of very variable thickness, ranging from a few decimeters to several hundred meters. Pillow lava structures have been recognized in several locations in the Combin zone (e.g.
Table 1 1 RSCM temperatures data See Additional file 1 for detailed measurements and calculations data. SR: Série Rousse; SG: Série Grise; TAR/C: Tracuit -Aig. Rouges zone / Cornet Unit; ES: Evolène Series 1 Retained measurements (spectra that could be fitted with r 2 >= 0.995) 2 Confidence interval (Student Law): 1α =
95% Series Sample nb Thin section nb Coordinates (MN95) Calculated temperatures (Beyssac et al. 2002) X Y Measur. nb. 1 Min. T [C] 1 Max. T [C] 1 STD (σ) 1 Conf. Int. 1,2 Mean T [C] 1 Hérens valley SR AP16035 S6-5 2′599′230 1′105′410 13 388 445 19 11 416 SR AP16036 S5-14 2′599′230 1′105′410 16 365 448 27 14 408 SR AP16055 S6-18 2′599′170 1′108′585 12 381 501 32 20 460 SR AP17007 S6-1 2′599′786 1′106′673 10 381 457 22 15 423 SR AP17017 S5-17 2′605′156 1′106′891 15 400 435 12 7 420 SR AP17040 S5-8 2′605′156 1′106′888 15 389 467 21 12 427 SR AP17C4 S6-10 2′605′303 1′107′142 12 396 486 24 15 423 SR AP1907 S7-6 2′603′082 1′105′740 13 411 473 21 13 439 SR AP2091 S9-3 2′602′061 1′108′060 13 380 458 18 11 414 SR AP2032 S9-5 2′605′443 1′104′767 11 431 513 23 16 467 SG AP17009 S5-15 2′605′004 1′106′290 16 397 458 18 10 436 SG AP17036 S5-9 2′605′228 1′106′869 13 412 507 29 17 466 SG AP19001 S7-1 2′605′385 1′103′175 15 388 507 32 18 435 SG AP19003 S7-5 2′604′862 1′104′049 15 424 495 23 13 446 SG AP1910 S7-3 2′602′498 1′105′612 13 417 465 15 9 442 SG AP1912 S8-14 2′603′410 1′105′465 14 416 511 26 15 450 SG AP2080 S9-2 2′604′400 1′103′220 12 391 451 17 11 411 TAR/C AP2083 S9-1 2′604′255 1′100′215 11 326 373 13 9 354 Bagnes valley SR AP1925 S7-2 2′594′349 1′094′318 12 437 537 33 21 483 SR AP1926 S9-6 2′594′217 1′094′242 11 436 541 31 21 459 ES AP15014 S1-17 2′591′951 1′095′923 14 418 549 31 18 484 ES AP2007 S8-7 2′592′654 1′094′496 13 433 541 34 20 480
|
10.1007/s43154-020-00028-z
|
cc-by
| null | null |
openalex
|
Purpose of Review The paper reviews the role of marine robots, in particular unmanned vehicles, in underwater surveillance, i.e. the control and monitoring of an area of competence aimed at identifying potential threats in support of homeland defence, antiterrorism, force protection and Explosive Ordnance Disposal (EOD). Recent Findings The paper explores separately robotic missions for identification and classification of threats lying on the seabed (e.g. EOD) and anti-intrusion robotic systems. The current main scientific challenge is identified in terms of enhancing autonomy and team/swarm mission capabilities by improving interoperability among robotic vehicles and providing communication networking capabilities, a non-trivial task, giving the severe limitations in bandwidth and latency of acoustic underwater messaging. Summary The work is intended to be a critical guide to the recent prolific bibliography on the topic, providing pointers to the main recent advancements in the field, and to give also a set of references in terms of mission and stakeholders' requirements (port authorities, coastal guards, navies).Introduction In the last decade, there has been a significant interest in the security of ports and marine infrastructures due to the growing threats against civilian targets. The vast scale of the European maritime region and the complexity of the challenges associated with ensuring successful maritime surveillance require a global approach to maritime security. As a result, projects involving different countries at European and NATO levels have been set up to fill the technological gap identified in this branch and to provide new methodologies. Underwater surveillance has traditionally been carried out by means of manned solutions. New maritime capabilities which include the way to conduct surveillance and protection of ports and coastal waters are treated within the Harbour & Maritime Surveillance and Protection (HARMPRO) project , implemented within the Permanent Structured Cooperation (PESCO) framework. In the same project framework, the Maritime Unmanned Anti-Submarine System (MUSAS) project will aim to develop an innovative system based on artificial intelligence for anti-submarine warfare that will strengthen the security of underwater infrastructures, providing a rapid reaction of sufficient measures of force to intruders. The Programme of Research for Defense Against Terrorism (DAT) provides measures to reinforce the organisation and coordination of actions to deal with terrorist incidents . This initiative includes programmes aimed at reducing the vulnerability of staff and facilities in all circumstances. Harbour operations are vital to the world economy and need to be as secure and safe as possible. For this reason, global institutions have been exploring various technologies to improve maritime security. In this framework, a key role is played by robotics, and more specifically by unmanned vehicles (UxV), that certainly are a game changing technology in this field. The involvement of UxV within a surveillance system helps in replacing human low level activities, giving to the human operator a high value information for higher decision level. Jointly working with other observation and manned systems, UxV are evolving into reliable, endurance, persistent and cost-effective surveillance solutions. Much has been debated for the use of these technologies, both at the scientific and operational level, highlighting the current potential possibilities and mapping a realistic outlook for their future. Such analyses are continually changing as, on the one hand, the technological development strengthens the roles already achieved in nowadays systems, making it possible to hypothesise more complex activities; on the other hand, scientific advancements provide creative potentials opening the way to new theoretical scenarios. The involvement of robotic systems in operating environments, whether military or civilian, has already begun to shift the methods that have historically been adopted in carrying out missions, such as detection, identification and surveillance. The Navy Unmanned Undersea Vehicle Master Plan is a valuable document that helps to map the employment scenario of the unmanned underwater vehicles (UUV), evaluating their capabilities and analysing their strengths, allowing for an accurate planning of acquisitions and technological research for many purposes. The Master Plan recognises the UUV component as a potential power multiplier in the operational scenario and a risk reduction tool for human operators. UUV capabilities allow end users to approach different task areas depending on the specific applications. The intelligence, surveillance and reconnaissance (ISR) abilities of UUVs can increase the capabilities currently running on systems historically operated by humans, expanding power and identification in complex environments, in high-risk areas for humans or even in shallow water where conventional systems are unable to operate. The relevance of the robotics impact on the field of maritime surveillance is also due to the capability of the UxV of hosting onboard, as payload, a variety of sensors as, e.g. sonars, hydrophones, magnetometers and optical or infrared cameras. Through these devices, the vehicles are able to conduct a rapid search task aimed at the identification and localisation of underwater targets and objects in confined areas, such as piers, within harbour areas or specific regions of interest. This paper offers a review of recent advancements in the role of robotics in underwater surveillance, i.e. the control and monitoring of an area of competence aimed at identifying potential threats in support of homeland defence, antiterrorism, force protection and Explosive Ordnance Disposal (EOD). The threats are asymmetric, and they can involve the use of surprises in both their tactical and strategic aspects, unpredictable behaviours and the use of no-conventional weapons in no-conventional ways. A revision of the recent literature on this topic is here proposed developing the discussion of previous related analyses . This work is to be intended as a critical guide to the recent prolific bibliography on the topic, giving also a general context in terms of mission requirements. The paper is organised in the following into two sections that address the involvement of robots in underwater surveillance under two complementary points of view: "Robots for Seabed Exploration" is related to use of robots for seabed surveillance, as typical in Mine Counter Measure (MCM) operations and analogous tasks; "Intrusion Detection Robots" is focused on the role of robots in systematic patrolling for intruder detection. "Main Research Frontiers in Underwater Surveillance," on the basis of the critical review of literature of the previous sections, proposes a discussion of the main research frontiers in underwater surveillance. "Conclusions" offers some general conclusions.
Robots for Seabed Exploration One core area of underwater surveillance includes the capability of identifying and localising explosive and hazard devices. This family includes improvised explosive devices (IEDs), naval mines and unexploded ordnance (UXO). The underwater IED represents a growing terrorist threat and is defined as an explosive device that is placed or fabricated in an improvised manner, by an irregular force created to hit a strategic or tactical goal. The IED risk is particularly high in Confined and Shallow Waters (CSW), especially in harbours, anchorages and routes to approach harbours. Underwater mines are defined as "explosive devices laid in the water by regular forces, with the intention of damaging or sinking ships or deterring shipping from entering an area" . They can be employed in a minefield or individually moored, floating, ground or buried. UXOs are old unexploded artefacts resting over or in the seafloor. They do not represent a real problem for already existing structures, but they must be taken into account before building a new infrastructure or in operation of clearing a new area. Clearing sea from UXO is a new challenging task for governments. A rich literature in UXO detection explains the multiple advantages of using magnetic sensors ; recently, considering the heterogeneous nature of UXO, there are relevant studies that consider also acoustic sensors and the reusage of already existing systems and algorithms for MCM purposes in order to perform UXO detection . Multiple and combined approaches can be used in order to contrast the mentioned threats, which can range from intelligence to simply selecting different routes during navigation. Surely, the inspection and the surveillance of the sea bottom are a fundamental step for an area to be considered safe. Traditionally, this has been done by using mine hunter/minesweeper vessels or deploying divers. Thus, man and expensive specific assets were used inside a potentially dangerous area. The usage of robots as UUVs has its main goal in reducing the risk for human operators and keeping them out of the minefield: no one knows how a mine is set and triggered; the only way to be safe is to stay far away from the explosive. Recently, the growing attention in artificial intelligence (AI) opened new scenarios in the usage of autonomous underwater vehicles (AUVs) . They can accomplish higher level tasks and take some decisions without the supervision of a human operator. This has a double meaning: on the one hand, it allows to cover the communication gap [11 ] that affects the underwater domain by limiting the amount of data that the vehicle requires to exchange; on the other hand, it allows to have a persistent control with the same level of attention [12, 13] without suffering from typical human stress conditions. UUVs can carry a plenty variety of sensors . These can range from acoustic ones, such as side scan sonar (SSS) and synthetic aperture sonar (SAS), which create a side-looking image of the sea bottom, to an echo sounder (Single and MultiBeam) that creates a 3D map. Sub Bottom Profiler (SBP) can also be used for the inspection of the sea bottom sedimentation. Furthermore, forward looking sonar (FLS) and acoustic cameras represent an alternative solution depending on the specific task. Magnetic sensors such as magnetometers and optical cameras and laser scanner may be used in very specific applications. Imaging sonar (SAS, SSS and FLS) operations using UUVs can employ image processing techniques to quickly and accurately find targets. Convolutional neural network (CNNs) can be considered the state-of-the-art performance of image classification tasks also in the maritime domain. Many authors propose their usage for both side-looking sonar [15, 16] and FLS achieving good results with well-trained networks.A considerable problem of the underwater domain is the difficulty of obtaining precisely geo-referenced images collected by a sensor and, consequently, the difficulty of subsequently locating found objects. The lack of GPS into the water makes necessary the use of solutions [18, 19] based on one or more surface gateways and systems of underwater positioning and communication such as ultra-short baseline (USBL), short baseline (SBL) or long baseline (LBL) techniques. While underwater localisation is offering promising results, a useful technique is the change detection (CD) method, based on the process of identifying objects or other phenomena of interest as temporal differences by observing a scene at different times . In mine hunting, CD is an important application. Reference sonar imagery of strategic ports, inlets or sea lines of communications are recorded during route surveys, when the seafloor is assumed to be free of mines. After a new survey, mines are recognised as objects that are only present in the current imagery reducing the possibility of false detection and the time to analyse data.
Intrusion Detection Robots Surveillance is one of the ways in which military and civilian implementations are very much intertwined and in several cases overlapping. These activities concern, for instance, coastal infrastructures, both military and civilian, including shipyards and offshore structures. Intruder threats can be divers or midget submarines. In addition, UxV should also be considered as a potential threat. Indeed, audio or image data collected by unmanned vehicles would be useful in strategic warfare against waterside facilities. Furthermore, they can potentially carry explosive payloads and may be used to target piers, merchant and military boats and docks , offering a significant advantage in asymmetric threat. Furthermore, UUVs could be potential intruders due to the smaller signature and target strength than the ones of a diver, and they are able to move faster, remaining below the detection threshold of active sonars at longer ranges . There are currently different kinds of surveillance systems and strategies under investigation. Since at least the early 1980s, various underwater security programmes have been implemented to counter the risks in ports . They mostly rely on the use of high-frequency active sonar, with detection ranges of less than 1 km. Traditionally, underwater surveillance has been conducted by manned platforms equipped with advanced and expensive sensors, which can be costly and with high workload . The UUVs can play a leading role in this context, cooperating with surface and underwater assets. The deployment of a team of unmanned surface vehicles (USV) to perform patrolling surveys may be very effective to improve the performance of a harbour security system . A solution to the issue of intercepting threats has been proposed by Simetti et al. based on the optimisation of interception time and the position by using a team of USVs conducting surveillance operations. Nardi et al. modelled as a potential game the issue of controlling a team of UUVs used as a defence system against asymmetrical threats, proposing a tool for designing the size of the team based on intruders' characteristics. In the H2020 WiMUST project framework , the design and experimental demonstration of a distributed reconfigurable autonomous underwater array has been addressed . The system consists of a team of cooperative robots equipped with hydrophone streamers for collecting seabed data, but it would support a significant range of applications, including acoustic surveillance. The use of cooperative robotics networks for underwater surveillance applications has been pursued by the NATO Centre for Maritime Research and Experimentation (CMRE). In the experiment presented by Ferri et al. , two cooperative UUVs equipped with acoustic sensors produced tracks onboard that were classified in real time and shared between the two UUVs and a ship acting as a high value asset (HVA), totally integrated into the network, in order to create a tactical picture. Since electromagnetic waves are strongly attenuated into the water, underwater surveillance based on acoustic waves becomes the most commonly used way to monitor the underwater environment. An example of underwater surveillance scenario is illustrated in Fig. 1 . Some solutions for localisation of underwater sources in surveillance applications employ acoustic vector sensors, which are rapidly growing in popularity and attention, since they can enhance the monitoring capabilities compared with the traditional hydrophones, thanks to their ability to provide directional information of noisy targets . Furthermore, they are small, lightweight, low power consumption and suitable to be used on UUVs: potentially, they can provide the same information of a towed hydrophone streamer through a compact sensor easy to install and deploy from a marine robot. The current drawback of vector sensors is the great sensitivity to robot motion, which has to be compensated in the raw data. The development of a dual accelerometer vector sensor mounted on the MEDUSA class AUV is described by Santos et al. . Although designed for geophysical surveys, the concept can be exploited for acoustic surveillance applications. The CMRE integrated a commercial vector sensor into the Slocum buoyancy glider , with the aim to demonstrate that UUVs can produce accurate target bearings. The obtained results potentially represent a starting point for the use of multiple gliders equipped with advanced sensors integrated into robotic surveillance networks. Experimental results on bearing estimation using a vector sensor from DIFAR sonobuoy mounted on the eFolaga hybrid AUV are presented by Terracciano et al. . The developed system shows very interesting performance in particular in terms of bearing estimation of multiple targets, providing an additional new tool for robotics underwater surveillance. Further approaches introduced include the use of volumetric arrays, as in the case of the CMRE which developed one for looking in all directions. It has been mounted on the nose of the Slocum glider to address the issue of marine traffic monitoring in a given maritime area .
Main Research Frontiers in Underwater Surveillance Considering the problems posed by underwater communications and networking, depicted in the previous sections, the latest research direction is to leave static and monolithic inbuilt modems and connectivity stacks for software-defined and cognitive, adaptable solutions. The use of Software-Defined Open-Architecture Modem (SDOAM) brings the great advantage of being able to calibrate all communication parameters (e.g. waveforms, frequencies, encodings) according to the conditions of the acoustic channel, which is strongly non-linear and variable . Recent works have shown how it is possible to adapt Fig. 1 A conceptual scenario of underwater surveillance based on unmanned systems. A network of UxV, each one carrying one or more specific payloads and fulfilling a specific mission, communicates, collects data and performs real-time monitoring. The communication infrastructure between the underwater and air environment is guaranteed by USVs acting as gateway buoys, i.e. equipped with acoustic and radio modems. The support ship is represented by NRV Alliance, a NATO research vessel used to conduct a wide range of oceanographic activities and that can act as C2S. In the sketch, UUVs are equipped with vector sensors and/or acoustic arrays for intruders' detection. Instead, some of them are equipped with sidescan sonar payloads for seabed exploration the operation of such modems according to requirements and vehicle mission profiles, taking into account specific channel metrics , even within a very short time of reaction . In order to use software-defined modems effectively, a cognitive and adaptable approach is also necessary in the upper layers of the communication protocol stack. In the recent past, several architectural solutions for networks have been proposed and developed that focus on fine synchronisation between the tasks performed by the various layers . These solutions have also been successfully implemented on AUVs and tested during sea trials [46, 47] . An example of a research centre that dedicated effort to the study, development and dissemination of this technology since 2011 is the CMRE. Recently, CMRE published an article outlining the main development and implementation activities of SDOAM and future directions . From this solid starting point, the CMRE communication stack is evolving into a fully cognitive communication architecture (CCA) to establish an intelligent, adaptable and safe underwater network . In addition, the scientific community, military end users and industry are increasingly paying attention to aspects relating to multi-vehicle operations, manned-unmanned interoperability and (cyber-)security . It is necessary to underline the fact that interoperability depends on many interconnected topics, such as the abovementioned Adaptive Autonomous Communications Networks, the Command and Control Systems (C2S) of robotics system-of-systems and the continuous standardisation of the interoperability. From the point of view of interoperability standardisation, there are two very significant NATO STANAGs which deserve to be listed in this paper: 4586 and 4748. First, STANAG 4586 is the current standard for unmanned aerial vehicles (UAVs) , which is the starting point for a new study aimed at establishing the "joint" multidomain control station (MDCS) standard, i.e. unmanned air, land and maritime systems. In the future, the MDCS Working Group-a combination of industry and government representatives-will formalise a revised version of STANAG 4586. Second, CMRE, together with academia and industry, has developed a standard underwater communication protocol known as JANUS , recently ratified by NATO as STANAG 4748 . This is the first internationally recognised and open-access underwater digital communication standard , already widely validated at sea for automatic identification system (AIS) data exchange and for the transfer of meteorological and oceanographic data to submarines . Thanks to the use of a standardised approach, it will be possible to increase the functional level of Maritime Situational Awareness (MSA) for robotic surveillance by using heterogeneous and hybrid systems from different manufacturers, as long as they comply with the JANUS standard. The fact that an underwater acoustic channel is inherently broadcast poses significant cyber-security risks in sensitive tasks, such as autonomous robotic surveillance, because an attacker is easily able to disrupt or intercept communications within such a network. The use of spread spectrum (SS) signals [59, 60] , as made in radio frequency (RF) domain, is one of the first possibilities of contrasting this problem. In contrast, the use of cryptographic keys in acoustic messages is not easily adaptable to the underwater environment. Therefore, an interesting concept is that of physical security , where the signal itself can be manipulated to generate confidential keys according to channel variability [62, 63] . A recent attempt to resolve the security issue at different levels of the network is addressed by Dini et al. , where the authors suggest a security framework specifically designed for underwater acoustic networks. This idea was implemented into the network deployed at sea in the context of the European project UAN . Finally, it is important to point out that a cooperative robotics network can use its own versatility and adaptability to respond to cyber threats as well [4, 66] . In this context, robotic surveillance systems are also required to be indefinitely persistent, regardless of the sea state, while maintaining a high level of effectiveness. Such a long-lasting use requires the development of innovative launch and recovery systems (LARS) systems and docking stations able to harvest energy 24 h a day at sea. All this involves a number of engineering problems that need to be tackled with special attention in the underwater field. The simple deployment and recovery operations of the surveillance vehicle team are often difficult and generally require a ship with adequate lifting and dynamic positioning (DP) systems. Solid, convincing and scalable engineering solutions for autonomous deployment, docking and recharging have to be pursued in order to ensure sufficient persistence and to relieve the workload of the support vessels [47, 67] . To date, one of the key challenges that researchers are facing in improving the persistence is the precise positioning of underwater vehicles, where the constraint of the drift of error over time becomes a crucial barrier to long-term missions as surveillance . Finally, lowering vehicle energy consumption by improving the efficiency of power supply and propulsion systems is essential if the duration of the mission would be extended from hours to days or even months in the case of gliders . A complementary approach is to recharge the internal batteries of the vehicle during the mission at offshore docking stations . A more flexible idea, almost unexplored so far, would be to equip the vehicle with a portable device capable of harvesting energy from the surrounding environment. For this reason, wave motion is especially appropriate because, theoretically, it is not limited by time and space, although the process of energy conversion is proving to be very difficult . This last line of research includes the Wave-powered Autonomous Vehicle for marine Exploration (WAVE) project [77, 78] whose final objective was to study, develop and test at sea a new wave energy harvesting and lowenergy propulsion system to be integrated into a generic, modular, torpedo-shaped AUV. In the SEALab framework , the joint laboratory between the Naval Support and Experimentation Centre (CSSN) and the Interuniversity Centre on Integrated System for the Environment (ISME), the authors outlined the recent advances on the use of maritime unmanned systems for military and dual-use applications.
Conclusions This paper provides a picture of the current state of the art about the involvement of robotic technologies within the underwater surveillance field of application. The attention is mainly focused on the impact that the use of unmanned vehicles had within this scope. These robots can host onboard a wide variety of sensors making them suitable to various typologies of operations; at the same time, the integration with the more traditional manned solutions and, in some cases, their partial substitution makes the unmanned vehicles a solution able to lower the overall operational costs. In addition to the versatility and the cost-effectiveness of the robot involvement, a great advantage of these technologies is their impact on human safety that drastically increases as the operators are shifted far from risky areas. These intrinsic advantages of robots make them gain more and more space in the operations typical of underwater surveillance. The paper provides a review of the recent examples, documented within the state of the art, of unmanned vehicles involved in two specific classes of operations typical of the underwater surveillance (Robots for seabed exploration, Section 2; Intrusion detection Robots, Section 3). The reader is critically guided through the recent bibliography on the topic, receiving references to the main recent advancements in the field and to sources about mission and stakeholders' requirements (port authorities, coastal guards, navies). The discussion of the current picture on the theme made possible to trace the road ahead towards greater and greater involvement of robots in the underwater surveillance field of application. These leading/primary research frontiers about underwater surveillance are widely discussed in the last section with references to the related bibliography. The main identified challenges include the autonomy enhancement and team/swarm mission capabilities to be achieved by the improvement of interoperability among robotic vehicles and by enabling communication networking capabilities, a non-trivial task considering the severe limitations in bandwidth and latency of acoustic underwater messaging. Funding Information Open access funding provided by Università di Pisa within the CRUI-CARE Agreement.
Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. 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/.
|
10.1016/j.ijscr.2019.03.032
|
cc-by
| null | null |
openalex
|
INTRODUCTION: Complete proper hepatic arterial [PHA] occlusion due to accidental coil migration during embolization of cystic artery stump pseudoaneurysm resulting from a complex vasculobiliary injurie [CVBI] post laparoscopic cholecystectomy [LC] is an extremely rare complication with less than 15 cases reported. We present a case depicting our strategy to tackle this obstacle in management of CVBI and review the relevant literature. PRESENTATION OF CASE: A 35 year old lady presented on sixth postoperative day with an external biliary fistula following Roux-en-y hepaticojejunostomy [RYHJ] for biliary injury during LC. She developed a leaking cystic artery pseudoaneurysm, during angioembolisation of which, one coil accidentally migrated into left hepatic artery resulting in complete PHA occlusion. Fourteen months later, cholangiogram revealed a worsening RYHJ stricture despite repeated percutaneous balloon dilatations. Multiple collaterals had developed. Revision RYHJ was fashioned to the anterior wall of biliary confluence with an extension into left duct. Minimum hilar dissection ensured preservation of collateral supply to the biliary enteric anastomosis. Postoperative recovery was uneventful. The patient is doing well at 1 year follow up. DISCUSSION: Definitive biliary enteric repair should be delayed till collateral circulation is established within the hilar plate, hepatoduodenal ligament and perihepatic/peribiliary collaterals to provide an adequate arterial blood supply to biliary confluence and extrahepatic portion of the bile duct. CONCLUSION: Assessment of hepatic arteries should be part of investigation of all complex biliary injuries. Delayed definitive biliary enteric repair ensures a well-perfused anastomosis. Minimum hilar dissection is the key to preserve biliary and hepatic neovasculature.Introduction Complex vasculobiliary injuries [CVBI] constitute 12-61% of biliary injuries, most commonly following laparoscopic cholecystectomy . They present a formidable challenge to the multidisciplinary team treating these patients. During management, complete right and left hepatic arterial occlusion due to accidental coil migration during embolization of a cystic artery stump pseudoaneurysm is an extremely rare complication [2, 3] . We present a case depicting our strategy to tackle this obstacle in the management of CVBI. This work has been reported in line with the SCARE criteria .
Case A 35 year old healthy lady presented to our department on sixth postoperative day [POD] with an external biliary fistula and intra-abdominal sepsis. She had undergone laparoscopic converted to open cholecystectomy for acute calculous cholecystitis. She had a biliary injury that was identified intra-operatively, managed by Roux-en-y hepaticojejunostomy [RYHJ] . The anastomosis leaked. An interno-external percutaneous transhepatic biliary drainage[PTBD] extending across the leak was performed at our hospital on POD 7 for both right and left hepatic ducts. On POD nine, she had an upper gastrointestinal bleed. Esophagogastroduodenoscopy and Contrast enhanced computed tomography [CECT] abdomen did not reveal the source of bleeding. On conventional hepatic arteriogram, a leaking cystic artery pseudoaneurysm was identified (Fig. 1 ). During angioembolisation, due to a short stump of cystic artery, coils were placed in right hepatic artery [RHA] . However, one of the coils accidentally migrated into the left hepatic artery [LHA] and could not be retrieved. LHA stenting was performed, with good flow of contrast across the stent (Fig. 2 ). However, LHA developed spasm in its distal part, resulting in complete block of LHA and RHA. On first day after coiling, there was significant elevation of liver enzymes with features of ischemic hepatitis. CECT abdomen with arteriography revealed poor enhancement of hepatic arterial tree in the segmental branches with partial revascularization from inferior phrenic and retroperitoneal arteries. The patient's relatives were explained the possibility of a need for an emergency liver transplant. The patient improved over the next 48 h, was transferred out of intensive care unit and oral feeds were started. Abdominal drain was removed after it stopped draining bile. She was discharged on POD 28 with PTBD catheters in situ. On POD 33, her liver function tests were within normal limits, percutaneous transhepatic cholangiogram showed trickle of contrast across the RYHJ, and the PTBD catheters were clamped. PTBD catheters were kept longer than 6 months and intervening periodic cholangiograms revealed anastomotic narrowing. Balloon dilatation of the stenosed anastomosis was performed on multiple occasions. Liver function was normal all along. Fourteen months after surgery, cholangiogram revealed a worsening of RYHJ stricture (Fig. 3 ). A CECT abdomen and conventional angiogram performed at 18 months showed a blocked RHA and LHA. Multiple collaterals were seen arising from the right inferior phrenic artery, retroperitoneum and along the hepatoduodenal ligament (Fig. 4 ). In view of the collateral formation and persistent tight stricture with failed multiple dilatations, a surgical revision of the anastomosis was planned. Prior to surgery, right PTBD catheter was maneuvered from right to left duct across the hilum, draining externally. During surgery, utmost care was taken not to release any adhesions in the hepatoduodenal ligament and not to mobilize liver from the diaphragmatic attachments to preserve all collaterals. Hilar dissection was kept to a minimum and the PTBD catheters guided the identification of biliary hilar confluence. A minor segement IV hepatotomy was done to expose the left hepatic duct and the roof of biliary confluence. Previous anastomosis was resected, and a Roux loop was prepared. A 2 cm single layer, interrupted 5-0 polydioxanone RYHJ was fashioned to the anterior wall of the biliary confluence with an extension onto the left duct. Her postoperative recovery was uneventful. Cholangiogram on POD 10 showed a good flow across the anastomosis with no sign of a leak, and she was discharged with a clamped PTBD catheter. The PTBD catheter was removed 6 weeks after the surgery. The patient is doing well at 1 year follow up. Liver function is normal. She is being followed up to evaluate for secondary patency.
Discussion CVBI, most commonly seen after a laparoscopic cholecystectomy, is defined as any biliary injury that involves the confluence or extends beyond it, any biliary injury with major vascular injury or any biliary injury in association with portal hypertension or secondary biliary cirrhosis . Major vascular injury is injury involving one or more of aorta, vena cava, iliac vessels, right hepatic artery, cystic artery, or the portal vein, seen in 0.04-0.18% of the operated patients and more so in patients with biliary injury . The vascular injury is suspected intraoperatively when there is significant bleeding during laparoscopic cholecystectomy, when there is a sudden rise in alanine aminotransferase during early postoperative course, or when there are multiple metallic clips on plain film images of the abdomen . The arterial injury may have been the result of the primary bile duct injury or may be the result of the attempted biliary repair [7, 8] . Vascular injury can present as pseudoaneurysm, usually within the first 6 weeks of post-operative period, abscesses over 1-3 weeks or ischemic liver atrophy after many years [2, 8] . Proper hepatic artery/both right and left hepatic artery involvement in CVBI has been very scarcely reported in literature. The reported cases in English literature and their management plan are presented in Table 1 [1, 7, . While these cases deal with intraoperative proper hepatic artery injury/occlusion, our case had a post RYHJ cystic artery pseudoaneurysm to begin with and the proper hepatic artery occlusion was an unfortunate event that took place during the embolization. There are occasional case reports of interventional proper hepatic artery pseudoaneurysm coiling leading to complete proper hepatic arterial occlusion with good outcome . However, a biliary injury complicated by cystic artery pseudoaneurysm bleed and then a proper hepatic artery occlusion has not been reported so far, to the best of our knowledge. Proper hepatic arterial injury induces biliary ischaemia and worsens a biliary injury. The hepatic ischemia is usually transient. The effect is more profound when the biliary confluence is disrupted along with a proper hepatic artery injury, since the hilar marginal collateral is also disrupted in these injuries . The effect on blood supply also affects the surgical outcomes. On univariate analysis in a study on factors affecting the surgical outcomes in biliary injury cases, VBI and sepsis were identified as factors for treatment failure. Also, 75% of these cases had complications and a poor long term patency rate . 5.6-15% of CVBI cases in another published series required liver resection. Known indications of hepatectomy included concomitant vascular injury and high biliary injury, liver atrophy, and intrahepatic bile duct strictures in their series [2, 8] . The clearing function of the liver with the translocated intestinal bacteria is impaired after ischemia and hence, it is important to maintain these patients with high antibiotic levels in the blood just to avoid septic complications in the ischemic liver parenchyma . Table 1 shows that these cases have a very high mortality rate and overall outcomes are poor. The management options include hepatic resection, liver transplantation in cases with fulminant liver failure and revision RYHJ with or without an arterial repair. Most of these cases have been managed by an early attempted RYHJ and arterial repair [1, 7, . However, temporary percutaneous biliary intervention to allow the collaterals to form has not been attempted in these cases. Since Michel's study on collateral circulation of liver to the present angiographic studies, it is now known that there are a lot of possible collateral channels to liver and bile ducts as shown in Fig. 5 . Collateral flow can be demonstrated 10 h after the occlusion. With time, the collaterals are good enough to sustain the biliary system . As seen in Fig. 5 , collaterals can develop from common hepatic artery, gastroduodenal artery, ligamentous arteries from falciform, coronary and right triangular ligaments, pancreatoduodenal arter- ies, the intercostal arteries and the phrenic arteries [14, 16] . In our case, the predominant collaterals were in the hepatoduodenal ligament and from superior and inferior phrenic arteries. Whilst waiting for the collateral supply to develop, the biliary anastomotic leak can be managed percutaneously by balloon dilatation and/or stenting as was performed in our case. Surgical planning involves identification of all collateral channels on the arteriogram. During surgery, liver mobilization has to be kept to minimum to preserve the ligamentous collaterals and hepatoduodenal ligament dissection also needs to be minimum . The revision anastomosis is performed with the standard surgical principles of RYHJ.
Conclusion Vascular assessment should be part of investigation of all complex biliary injury cases. Delayed definitive repair in cases involving PHA occlusion to allow collateral circulation to be established within the hilar plate, hepatoduodenal ligament and perihepatic/peribiliary collaterals and thereby provide an adequate arterial blood supply to the biliary confluence and the extrahepatic portion of bile duct is a feasible management option. Minimum dissection should be done during surgery to preserve biliary and hepatic neovasculature.
Conflicts of interest Authors declare that they have no conflict of interest.
Sources of funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Ethical approval Institutional ethics committee approval was taken for the publication.
Consent Informed consent was obtained from the patient for this publication.
Author's contribution Gunjan Desai: collecting data, analysis of data, preparing the initial draft of the manuscript, critical revision of the manuscript for intellectual content, technical support, material support, study supervision. Prasad Pande: critical revision of the manuscript for intellectual content, technical support, material support, study supervision. Rajvilas Narkhede: critical revision of the manuscript for intellectual content, technical support, material support, study supervision. Dattaprasanna Kulkarni: study concept, critical revision of the manuscript for intellectual content, administrative, technical support, material support, study supervision.
Registration of research studies This is a case report and does not require such registration. The publication was performed after the approval of research protocols by the Ethics Committee of Lilavati Hospital and Research Centre in accordance with international agreements (World Medical Association Declaration of Helsinki "Ethical Principles for Medical Research Involving Human Subjects," amended in October 2013, www.wma. net).
Guarantor Gunjan S Desai is the article guarantor.
Provenance and peer review Not commissioned, externally peer-reviewed. Fig. 1 . 1 Fig. 1. Conventional celiac artery angiogram showing the cystic artery pseudoaneurysm[yellow arrow].
Fig. 2 . 2 Fig. 2. Conventional celiac artery angiography and coiling of cystic artery pseudoaneurysm and right hepatic artery and coil migration into left hepatic artery followed by stenting of the left hepatic artery[yellow arrow].
Fig. 3 . 3 Fig. 3. Percutaneous transhepatic cholangiogram showing the Roux-en-y hepaticojejunostomy anastomotic stricture.
Fig. 4 . 4 Fig. 4. Conventional celiac artery angiogram showing collaterals[red arrows] from the proper hepatic artery, inferior phrenic artery and along the hepatoduodenal ligament.
G .S. Desai et al. / International Journal of Surgery Case Reports 58 (2019) 6-10
Fig. 5 . 5 Fig. 5. Schematic diagram to show the possible arterial collateral channels[numbered dotted lines] to the liver. SEA: Superior epigastric artery, MPA: Musculophrenic artery, ASPA: Accessory superior phreinc artery, ICA: Intercostal artery, AB: Anterior branch, PB: posterior branch, LB: Left branch, RB: Right branch, BPA: Branch to phrenic artery,IPA: Inferior phrenic artery, AIPA: Accessory inferior phrenic artery, RTL: Right triangular ligament, LTL: Left triangular ligament, FA: Falciform artery, RHA: Right hepatic artery, MHA: Middle hepatic artery, LHA: Left hepatic artery,CA: Celiac artery, LGA: Left gastric artery, CHA: Common hepatic artery, SPDA: Superior pancreaticoduodenal artery, PHA: Proper hepatic artery, GDA: Gastroduodenal artery, RDA/SDA: Retroduodenal artery/Supraduodenal artery, LGE: Left gastroepiploic artery, CPA: Caudal pancreatic artery, APMA: Arteriapancreatica magna, DP: Dorsal pancreatic artery, TP: Transverse pancreatic artery, AE: Anterior epiploic artery, PE: Posterior epiploic artery, RGE: Right gastroepiploic artery, A/R LHA: Accessory/Replaced left hepatic artery, A/R RHA: Accessory/Replaced right hepatic artery, RCHA: Replaced common hepatic artery, ATPA: Accessory transverse pancreatic artery, SMA: Superior mesenteric artery, IPDA: Inferior pancreaticoduodenal artery, LE: Left epiploic artery, RE: Right epiploic artery.
Table 1 1 Reported cases of complex vasculobiliary injuries involving common hepatic artery or proper hepatic artery during cholecystectomy, their presentation, management and outcomes. Sr no. Year Arterial injury Biliary injury Identification and Presentation Management Follow-up Outcome 1 Frilling et al. Proper hepatic artery E2 During first surgery End-to-end repair of artery and None Death on POD 28 - Roux-en-Y hepaticojejunostomy - could not undergo liver On POD 16, re-exploration showed transplant ligated right hepatic artery and thrombosis of superior mesenteric vein and portal vein -right hepatectomy and listed for urgent liver transplantation 2 Frilling et al. Common hepatic artery E1 POD 17 Reconstruction of artery using None Death on POD 37 mesenteric venous graft and external drainage of both biliary ducts -Roux-en-Y hepaticojejunostomy after 3 days 3,4 Wudel et al. Common hepatic artery Not available Not available Not available Not available Not available 5 Gupta et al. Proper hepatic artery Strasberg E4 Postoperative day 4 Bilateral hepaticojejunostomies One and half year + Recovered with without vascular intermittent biliary repair/reconstruction strictures being managed with percutaneous transhepatic balloon dilatations 6 Buell et al. Common hepatic artery Transected right Average 8.5 days Primary repair of artery and None Death hepatic duct percutaneous external biliary drainage 7 Buell et al. Common hepatic artery Major biliary injury - Average 8.5 days Right hepatectomy and posted for None Death Roux-en-Y liver transplantation hepaticojejunostomy in first surgery 8 Buell et al. Common hepatic artery Major biliary injury - Average 8.5 days Orthotopic liver transplantation Regular Alive Roux-en-Y hepaticojejunostomy in first surgery First surgery was open Second surgery on postoperative 9 Salman et al. Proper hepatic artery E3 cholecystectomywith day 3 -T-tube drainage and One year + Recovered right hepatic artery hemostasis ligation Percutaneous drainage of cholangitic liver abscess on POD 85 Third surgery on postoperative day 144 -Roux-en-Y hepaticojejunostomy 10 Yan et al. Proper hepatic artery E4 Roux-en-Y Secondary biliary cirrhosis and Range of 4-17 months Recovered hepaticojejunostomy during portal hypertension on follow up first surgery{laparoscopic {Range of 5-148 months} - cholecystectomy} or first Underwent liver transplantation postoperative week of surgery without repair/reconstruction of artery 11 Strasberg et al. Proper hepatic artery Necrosis of the Postoperative day 6 with a Liver transplantation on None Death on post intrahepatic biliary large biliary fistula Postoperative day 39 transplant day 14 tree till fourth order branches 12, 13 Sarno et al. Common hepatic artery Major biliary injury Details not available Primary or revisional Follow up range of Recovered hepaticojejunostomy. Vascular 12-245 months repair/reconstruction not described
|
10.1155/2017/4068963
|
cc-by
| null | null |
openalex
|
Purpose. To evaluate changes in anterior, posterior, and total corneal astigmatism in patients after Descemet membrane endothelial keratoplasty (DMEK). Methods. We retrospectively included 29 eyes of 23 patients (age 67.6 ± 9.8 years, 13 female, 10 male) after DMEK surgery. The magnitude and axis orientation of anterior, posterior, and total corneal astigmatism before and after DMEK were determined using a rotating Scheimpflug system (Pentacam HR, Oculus). Results. The magnitude of anterior, posterior, and total corneal astigmatism in the central cornea did not change significantly after surgery. Before surgery, we found a significant correlation between the magnitudes of anterior and posterior corneal astigmatism (Spearman's correlation coefficient r S = 0 526, P = 0 003), while after surgery this correlation was no longer significant (r S = 0 038, P = 0 843). There was a significant correlation between the vector difference between preoperative and postoperative posterior astigmatism and the change in corneal pachymetry (r P = 0 47, P = 0 010). Conclusions. Posterior corneal astigmatism (especially the orientation) and therefore the relationship between anterior and total corneal astigmatism may change after DMEK. This should be considered to improve the accuracy of toric IOL power calculations following phakic DMEK or in combined procedures.Introduction With advantages such as a lower risk of rejection, faster visual rehabilitation, improved visual outcome, and refractive stability after DMEK , DMEK surgery is becoming an increasingly popular option for the treatment of Fuchs' endothelial dystrophy (FED) [6, 7] . Cataract surgery can be performed together with DMEK in the same setting , or in phakic eyes after DMEK surgery . Cataract surgery and implantation of toric intraocular lenses (IOLs) are often performed to correct corneal astigmatism, resulting in satisfactory spectacle-free visual outcomes. To achieve perfect correction of corneal astigmatism, correct and precise measurements of corneal astigmatism must be obtained to determine the magnitude and meridian . Wacker et al. showed relevant changes in corneal astigmatism based on asymmetric corneal swelling in the course of FED and demonstrated differences compared with the normal population at different stages of the disease . During the new triple procedure with toric IOL implantation, the corneal shape, especially of the posterior corneal curvature, can be expected to change [5, 11] . This may affect the accuracy of toric IOL power calculations. The study presented here aims to assess the changes in magnitude and axis orientation of anterior, posterior, and total corneal astigmatism by means of the Pentacam Scheimpflug imaging system (Oculus, Wetzlar, Germany) in patients with FED after DMEK surgery.
Materials and Methods This retrospective study included 29 eyes of 23 patients who underwent DMEK surgery at the Dept. of Ophthalmology, University of Muenster Medical Center. The study was prospectively approved by the local ethics committee and adhered to the tenets of the Declaration of Helsinki. Patients were examined after attaining refractive stability (3-12 months after surgery) [5, 12] using rotating Scheimpflug corneal and anterior segment tomography (Pentacam HR; Oculus, Wetzlar, Germany). Only patients with complete pre-and postoperative Pentacam data with good quality corneal tomography scans were included. A skilled examiner performed Pentacam imaging; the automatic release mode of the Pentacam was used to minimize examiner-induced errors, and all patients were examined under the same conditions. Eyes with a history of other corneal diseases, corneal infection or intraocular inflammation, trauma, corneal scars, contact lens wear four weeks before measurement, clinically significant graft detachment, or delayed corneal clearance were excluded. Some of the subjects had been included in our previous reports on refractive evaluation of patients with FED and patients after endothelial keratoplasty . Corneal pachymetry at the apex and corneal topography data including average keratometry reading (Km F ), average keratometry reading of the posterior corneal surface (Km B ), and the magnitude and axis orientation of anterior, posterior, and total corneal astigmatism on the central 15 ring (equal to the 3.0 mm ring) were obtained and analyzed. (1) Anterior corneal astigmatism (AA) is the astigmatism value arising from the anterior corneal surface alone and calculated as the difference in simulated keratometry values (calculated using the standard keratometric index (1.3375) and the radius of anterior corneal curvature) between the steepest and the flattest meridians . (2) The posterior corneal astigmatism is defined as the difference in keratometry values between the flattest and steepest meridians, which were calculated using the radius of posterior corneal curvature and the refractive indices of the cornea (1.376) and aqueous humor (1.336) . (3) Total corneal astigmatism is defined as the difference in total corneal refractive power between the steepest and flattest meridians (calculated by ray tracing through the anterior and posterior corneal surfaces, according to Snell's law) . For analysis of the relative orientations of corneal astigmatism, anterior corneal astigmatism, and total corneal astigmatism, these were classified as follows : (1) with-the-rule: 60 < the steep meridian < 120 (2) against-the-rule: 0 < the steep meridian < 30 or 150 < the steep meridian < 180 (3) oblique: otherwise. Since the dioptric power of the posterior corneal surface is negative, the posterior corneal astigmatism was classified as follows : (1) with-the-rule: 0 < the steep meridian < 30 or 150 < the steep meridian < 180 (2) against-the-rule: 60 < the steep meridian < 120 (3) oblique: otherwise. The vector difference between preoperative and postoperative posterior corneal astigmatism was calculated by vector analysis, as described by Holladay et al. . All patients underwent standard DMEK surgery (between 2014 and 2015), performed by the same surgeon (LZ). The surgical procedure was described previously in details [1, 11] . 2.1. Statistical Analysis. Data management was performed with Microsoft Excel 2010. IBM SPSS Statistics 22 for Windows (IBM Corporation, Somers, NY, USA) was used for statistical analyses. The normality of the data distribution was tested using the Kolmogorov-Smirnov test. A normal distribution of the data was confirmed for Km F , anterior corneal astigmatism, total corneal astigmatism, Δ pachymetry at the apex, and the vector difference between preoperative and postoperative posterior corneal astigmatism. Parametric analysis (Student's t-test for paired values, Pearson's correlation coefficient (r P )) was then carried out. When the data did not fit a normal distribution (Km B , posterior corneal astigmatism), nonparametric tests (two-sided Wilcoxon signed-rank test, Spearman's correlation coefficient (r S )) were performed. Changes at follow-up compared with baseline were assessed, assuming left and right eyes of the same patient to be independent. Data are reported as mean ± standard deviation. The level of statistical significance was set at P <= 0 05. Inferential statistics are intended to be exploratory, not confirmatory, and were interpreted accordingly.
Results The demographics of our population are summarized in Table 1 . The corneal thickness at the apex and average keratometry reading of the posterior corneal surface (Km B ) changed significantly after surgery (P < 0 001). The magnitudes of anterior, posterior, and total corneal astigmatism did not change significantly after DMEK (anterior astigmatism: before =1.14 ± 0.76 D, after =1.30 ± 0.77 D, P = 0 43; posterior astigmatism: before =0.38 ± 0.29 D, after =0.33 ± 0.20 D, P = 0 76; total astigmatism: before 1.64 ± 1.33 D; after =1.61 ± 0.90 D, P = 0 93). ATR astigmatism of the posterior corneal surface was found in 27.59% (n = 8) of patients before surgery and in 86.21% (n = 25) of patients after DMEK surgery. Figures 1(a ) and 1(b) show the orientation of anterior, posterior, and total corneal astigmatism before (a) and after (b) DMEK surgery. The proportion of WTR astigmatism in anterior and total corneal astigmatism before and after surgery did not change markedly (Figure 1 ). Figure 2 shows the location of the steep meridian on anterior and posterior corneal surfaces before and after DMEK surgery. Before surgery, we found significant correlations between the magnitudes of anterior and posterior corneal astigmatism (r S = 0 53, P = 0 003), between those of posterior and total corneal astigmatism (r S = 0 47, P = 0 010), and between those of anterior and total corneal astigmatism (r P = 0 85, P < 0 001). After surgery, the correlations between the magnitudes of anterior and posterior corneal astigmatism and between those of posterior and total corneal astigmatism were not significant, (r S = 0 04, P = 0 843) and (r S = -0 07, P = 0 721), whereas we found a strong correlation between the magnitudes of anterior and total corneal astigmatism (r P = 0 92, P < 0 001) (Figure 3 ). Figure 4 shows the changes in the relationship of anterior to total corneal astigmatism before and after DMEK surgery and underlines the complexity of accurate estimation of posterior corneal astigmatism based 3 Journal of Ophthalmology on the measurement of the anterior corneal curvature in these cases. Figure 5 shows the doubled-angle plot for the vector difference between the posterior corneal astigmatism before and after DMEK. Moreover, there was a significant correlation between the vector difference between preoperative and postoperative posterior corneal astigmatism and the change in corneal pachymetry at the apex (r P = 0 47, P = 0 010).
Discussion Toric IOLs have been developed to reduce corneal astigmatism at the time of cataract surgery. Owing to the excellent visual outcome and refractive stability achieved after DMEK [5, 12] , (toric) IOL power calculations in patients after phakic DMEK or within the setting of the new triple procedure are becoming a topic of much interest in clinical practice [18, 19] . As previously described, the change in corneal Figure 3 : Showing correlations between the magnitudes of anterior posterior and total corneal astigmatism before (blue) and after (red) DMEK surgery. Before surgery, we found a significant correlation between the magnitudes of anterior and posterior corneal astigmatism (Spearman's correlation coefficient r S = 0 53, P = 0 003); after surgery, this correlation was not significant (r S = 0 04, P = 0 843).
4 Journal of Ophthalmology refractive power after DMEK is based mainly on changes in the posterior corneal curvature, whereas the anterior corneal curvature does not alter significantly [5, 11] . The importance of posterior corneal astigmatism has recently been highlighted in various studies . Toric IOL power calculations based on the anterior corneal curvature, ignoring posterior corneal astigmatism, can over-or underestimate total corneal astigmatism . Different studies have shown that with-the-rule is the most common type of anterior corneal astigmatism among younger individuals, whereas against-the-rule astigmatism becomes more common with aging [14, 25] . Posterior corneal astigmatism remains ATR with aging [14, 25] . In this study, our results demonstrate that the mean magnitudes of anterior and total corneal astigmatism did not change significantly after DMEK surgery. There was no significant change in the magnitude of posterior corneal astigmatism either. Our results are in line with previous findings concerning the magnitude of posterior corneal astigmatism in normal eyes and in eyes with FED [10, 13, 16, 25, 26] . We know that in eyes with WTR astigmatism of the anterior corneal surface, the presence of ATR astigmatism of the posterior corneal surface compensates for anterior corneal astigmatism and thus reduces total corneal astigmatism. However, in eyes with ATR astigmatism of the anterior corneal surface, it increases total corneal astigmatism [15, 26] . Wacker et al. showed that eyes with advanced FED were more likely than normal eyes to have oblique or WTR posterior corneal astigmatism . In this study, we found that the posterior corneal surface shifts back from with-the-rule to against-the-rule astigmatism after surgery in most cases, whereas no marked change was observed in the orientation of the anterior or total corneal astigmatism. The vector difference between preoperative and postoperative posterior corneal astigmatism correlates with the change in corneal thickness. These findings will be helpful for calculation of toric IOL power within the setting of the triple procedure. Yokogawa et al. demonstrated in small case series (10 patients) that triple DMEK procedure with toric IOL implantation provided relatively good UDVA and reduced refractive astigmatism. However, compared with simple cataract surgery with toric IOL implantation, the postoperative corneal astigmatism may not be as predictable as in patients undergoing cataract surgery alone . This study and our results demonstrate that the calculation of toric IOL power in the setting of new triple procedure is complicated by the expected changes in corneal curvature. In cases with minimal or mild preoperative corneal swelling, it is advisable, given the impact of posterior corneal astigmatism , to acquire extensive biometric data from the entire cornea and consider the orientation and magnitude of a measured 5 Journal of Ophthalmology posterior corneal astigmatism and to factor in the changes demonstrated here. For assessment for the new triple procedure in cases with severe preoperative corneal swelling, it would be difficult to predict changes in corneal astigmatism. The large number of outliers, as shown in Figure 4 , should be taken into account when deciding whether to plan a toric IOL implantation or rather, in critical cases, to go with a standard IOL. The other possible way is to perform phakic DMEK and then to perform cataract surgery after corneal stabilization; in this case, the risk of endothelial cell loss caused by secondary cataract surgery has to be taken into account. For more accurate prediction of corneal astigmatism after DMEK, the deswelling profile of the cornea and factors that might influence these changes needs to be evaluated in detail. For calculation of toric IOL power after phakic DMEK, given the difference in magnitude and axis orientation between anterior and total corneal astigmatism seen in our population (Figure 4 ), we recommend a calculation based on a measured anterior and posterior corneal curvature after attainment of refractive stability, compared with population-averaged value. In our opinion, this would improve the accuracy of toric IOL power calculations in these particular patients. In patients with FED, we found a significant correlation between the magnitude of the anterior and posterior as well as posterior and total corneal astigmatism at the steepest corneal meridians. This correlation was comparable to findings previously described for normal or keratoconic corneas , suggesting that the role of the posterior cornea may be more critical in cases with a high anterior or total corneal astigmatism. After DMEK, the correlations between posterior/anterior and between posterior/total corneal astigmatism were not significant. Furthermore, the correlation coefficient between anterior and total corneal astigmatism after surgery was higher than before. This study is burdened with some limitations, which may have affected the results to some extent. First, the follow-up time after surgery (3-12 months) was not exactly defined. However, refractive stability for 3 months after DMEK has been described previously in the literature [5, 12] . Second, it was performed in a retrospective design. Third, we included a relatively small number of eyes. However, further studies in a prospective setting with detailed long-term analysis, especially of the posterior corneal curvature before and after DMEK, are urgently needed. In conclusion, posterior corneal astigmatism should be considered for more accurate total corneal astigmatism predictions. It should be noted that the orientation of the posterior corneal astigmatism and the relationship of anterior to total corneal astigmatism appear to change after DMEK surgery, whereas the magnitude of the posterior corneal astigmatism does not change significantly. The vector difference between preoperative and postoperative posterior corneal astigmatism correlates with the change in corneal thickness. Finally, the essential point to emphasize is that the power of astigmatism may change as a result of DMEK surgery. However, attention should also be paid to possible changes in axis orientation. These simple findings are clinically very important for the calculation of toric IOL power within the setting of the new triple procedure and in patients after phakic DMEK. Figure 1 :Figure 2 : 12 Figure 1: Distributions of anterior, posterior, and total corneal astigmatisms before (a) and after DMEK surgery (b). ATR: astigmatism against-the-rule; WTR: astigmatism with-the-rule; oblique: astigmatism oblique.
Figure 4 : 4 Figure4: Showing the differences in magnitude and orientation between anterior and total corneal astigmatism before (blue) and after (red) DMEK. The figure illustrates the complexity of the estimation of total corneal astigmatism based on anterior corneal curvature. Note the large number of outliers.
Figure 5 : 5 Figure 5: Doubled-angle plot for the vector difference between the posterior corneal astigmatism before and after DMEK. The red spot shows the centroid.
Table 1 : 1 Demographics of the study population in eyes after Descemet membrane endothelial keratoplasty. Mean ± standard deviation (minimum-maximum). Bold: statistically significant results. Before surgery After surgery P value
Journal of Ophthalmology
|
10.1186/s12913-017-2156-9
|
cc-by
| null | null |
openalex
|
Background: We assessed the 30-day risk of readmission and mortality among patients receiving an International Classification of Diseases 10th edition diagnosis of medical observation and evaluation (Z03*) following admission to an acute medical admission unit (AMAU), stratified on any further specification of diagnosis during hospital stay. Methods: We used Central Denmark's (Midt)-Electronic Patient Journal to identify patients with a Z03diagnosis among patients admitted to the AMAU, Aarhus University Hospital Nørrebrogade from April 2012 to March 2013, and noted any specification of diagnosis. Patients were followed from hospital discharge until death, emigration, or completion of 30 days follow-up. Results: Of 409 patients with an initial Z03* diagnosis at the AMAU, 55% (n = 226) received a more specific discharge diagnosis after transferral to other departments. Among patients discharged to home with a Z03diagnosis, 30% were readmitted within 30 days, while the corresponding figure was 23% for patients receiving a specific diagnosis (p = 0.06). In contrast, corresponding figures for 30-day mortality were 3% for Z03diagnosed patients and 10% for those who obtained a specific diagnosis (p = 0.003). Conclusions: Patients diagnosed with Z03* at hospital discharge have a substantially lower 30-day mortality, but a higher readmission-rate, compared to patients who obtain a specific diagnosis during the entire hospital stay.Background Health care systems now face an ageing population with increasing multimorbidity in Denmark and elsewhere. It can be expected that an increasing proportion of acutely hospitalized medical patients will present with advanced age and comorbidities in the future . Previous research shows that non-specific diagnoses, including ill defined (Rcoded) conditions and encounters for medical observation for suspected diseases and conditions (Zcoded) are highly prevalent in patients in Denmark and other countries including Iceland and the UK, amounting to approximately 20% of all unplanned or emergency admissions . Zdiagnoses (85% of these being Z03*; "Medical observation and evaluation for suspected diseases and conditions" from chapter XXI in the International Classification of Diseases, Tenth Revision (ICD-10)) currently account for 17% of all patients admitted acutely to Danish medical departments [2, 5] and the use of Zdiagnoses appears to be increasing . Z03diagnoses include, quoting the World Health Organization (WHO) "persons who present some symptoms or evidence of an abnormal condition which requires study, but who, after examination and observation, show no need for further treatment or medical care" . In a previous study covering acutely admitted patients in Denmark, Vest-Hansen et al. , observed that Z03coded patients were equally divided by gender, the majority was aged 60-79 years, and the majority did not have major comorbidities before the index admission. This indicates that these patients may be generally relatively healthy at baseline, compared to other hospitalized patients in this age group . The large proportion of patients receiving unspecific diagnoses in Denmark and elsewhere underscores the need of further examination of this patient group and their clinical course and prognosis. Thus, the aim of our study was to examine 30-day readmission risk and 30-day post-discharge mortality among all patients discharged from an acute medical admission unit with a Z03diagnosis, and whether it differed if the patient received further specification of diagnosis during hospital stay or not.
Methods
Study setting and design We conducted this study in one hospital in Aarhus, Denmark (population by April 1st 2012, N = 289.941 ). In Denmark's tax-supported healthcare system, all citizens have equal access to specialist treatment and hospital care . All Danish citizens receive a unique personal identification number (CPR number) at birth or immigration, used in all health databases and permitting unambiguous record linkage . We created a cohort of all patients with a Z03 diagnosis, using Danish medical registries, which we followed with respect to mortality, re-hospitalization or end of follow-up.
Study population The study population included all Danish citizens aged over 18 years admitted to the AMAU, Aarhus University Hospital Nørrebrogade, Aarhus, Denmark, between April 1st 2012 and March 31st 2013 with a primary diagnosis of Z03* at discharge from AMAU, as registered in the Midt-Electronic Patient Journal (MidtEPJ). The MidtEPJ is a clinical care tool, and is related to an underlying patient administration system (PAS), which contains administrative data on every hospital contact, including time of admission, department, temporary location, and primary and secondary discharge diagnoses classified according to the International Classification of Diseases, Tenth Revision (ICD-10) since 1994 [7, 11] . Core data from the PAS are forwarded to the Danish National Patient Registry (DNPR). The MidtEPJ contains essential data not forwarded to DNPR, including temporary location codes, which in this study were crucial to identify patients at the AMAU. Medical patients with suspicion of selected conditions (acute myocardial infarction (AMI), stroke, or pregnancyrelated conditions) are referred directly to highly specialized departments, instead of being first admitted to AMAU.
Classification of length of stay, departmental transfers, and specification of diagnoses The length of an AMAU stay was classified as the time period in hours from AMAU admission to AMAU discharge, whether the discharge was directly to home or to another department (Fig. 1 ). The length of entire hospital stay was defined as the period from time of AMAU admission to the time of final hospital discharge, from AMAU or another department. We considered the patient discharged from hospital, if the time period between a department discharge and a new department admission exceeded 24 h. Obtainment of a specific diagnosis during the entire hospital stay was defined as an initial Z03diagnosis achieved at AMAU discharge together with a non-Z03*diagnoses achieved at final discharge from any department during further hospital stay.
Readmission and mortality We followed the patients from the date of hospital discharge (entire hospital stay) until death, emigration, or completion of 30 days of follow-up. Vital status was obtained through the Danish Civil Registration System (CRS), which includes exact date of death, updated daily . We registered any readmission within 30 days after hospital discharge, using MidtEPJ.
Comorbidity Using information from PAS, we obtained information on the patient comorbidity burden at AMAU admission using the Charlson Comorbidity Index (CCI) . We computed the CCI-score using all preceding primary diagnoses up to 5 years before the date of AMAU admission. We divided CCI-scores into low level (Index score 0), moderate level (Index score 1-2) and high level (Index score 3+). The CCI has been shown to have a high predictive ability in Danish medical registries .
Statistical analysis We described characteristics of the AMAU population. We estimated the median length of the stay at the AMAU and the entire hospital stay, and calculated the proportion of patients discharged to home, or with transferrals to another department. We calculated the proportion obtaining a specific diagnosis during the entire hospital stay. We calculated the proportion of all patients readmitted within 30 days from hospital discharge, stratified by whether patients had obtained a specific diagnosis or maintained Z03diagnoses. We computed 30-day mortality overall and stratified by whether the patients obtained a specific diagnosis after transferral or maintained Z03*diagnoses. We compared the proportions readmitted and 30-day mortality for patients obtaining a specific diagnosis or maintained the Z03diagnosis, using a one-sided ztest, with a significance level of 0.05. Data were analyzed using SAS version 9.4 According to Danish legislation, individual informed consent or additional permission from the National Committee on Health Research Ethics is not required for studies based on information from registries and electronic databases with no patient contact taking place. In Denmark, a project, such as this being classified as an internal clinical quality enhancement project can gain access to data after being approved by the management of the clinical department, as this was.
Results Of the 4,916 patients admitted to AMAU, 8% (409) were discharged from AMAU with a Z03diagnosis. Median age of these patients was 69 years (interquartile range (IQR) 56-80 years), 51% were male, and 55% had no comorbidity (CCI-score = 0) (Table 1 ). The majority of patients (76%) were diagnosed with either "Observation for suspected disease or condition, unspecified" (Z03.9) or "Observation for other suspected diseases and conditions" (Z03.8).
Length of hospital stay, diagnoses, internal transfers The median length of the AMAU stay was 14 h (IQR 6-21 h) and the median length of the entire hospital stay was 75 h (IQR 17-187 h). Figure 2 shows the flow of patients from admission to discharge, and subsequent readmission and diagnoses. Of 409 patients with Z03diagnoses, 34% (138) were discharged directly to home from the AMAU. The remaining 271 patients were transferred, and 83% (226/271) obtained a specific diagnosis, corresponding to 55% (226/409) of all individuals with an initial Z03diagnosis at the AMAU. The most common diseases among the 226 patients who eventually obtained a diagnosis were within ICD chapter J (respiratory diseases = 21%, most commonly pneumonia, COPD, or respiratory failure), chapter K (digestive diseases = 15%, most commonly alcoholic liver disease, cholelithiasis, or hepatic failure), chapter I (circulatory diseases = 12%, most commonly cerebral infarction, heart failure or atrial fibrillation and flutter), chapter C (malignant neoplasms = 11%, most commonly malignant neoplasm of bronchus and lung, or malignant neoplasm of pancreas) and chapter A (infectious and parasitic diseases = 10%, most commonly sepsis, gastroenteritis and colitis, or erysipelas).
Readmission Of the 138 patients discharged directly to home, 28% (38/138) were readmitted within 30 days after hospital discharge (Fig. 2 ). After readmission, 24% (9/38) were discharged with Z03diagnoses, while 76% (29/38) obtained a specific diagnosis. Of the 45 patients who maintained Z03diagnoses after transferral, 3 (6%) died during the hospitalization. Of the 42 patients eligible for readmission, 38% (16/42) were readmitted, and 69% (11/16) of these patients were discharged with a specific diagnosis. Fig. 2 Proportion of all readmitted within 30 days from hospital discharge, stratified on maintaining diagnosis or further specification Thus, among all patients with a Z03diagnosis at hospital discharge, 30% (54/180) [95% CI 23.8;37.0] were readmitted within 30 days. Among the 226 patients who obtained a specific diagnosis after transferral, 14% (31) died during further hospitalization. Of the 195 eligible for readmission, 23% (45/195) [95% CI 17.7;29.5] were readmitted, and here 93% (42/45) obtained a specific diagnosis. We observed a tendency towards a higher proportion being readmitted in the patients with Z03diagnosis at discharge compared to patients who obtained a specific diagnosis (p = 0.06).
Mortality The overall mortality during entire hospital stay and subsequent follow-up period was 14% (59/409) (Fig. 3 ). No patients died in the AMAU. Among all patients with a Z03diagnosis at hospital discharge, the 30-day mortality was 3% (5/180) [95% CI 1.2;6.3]. Among the patients who obtained a specification of diagnosis during the entire hospital stay, the 30-day post-discharge mortality was 10% (20/195) [95% CI 6.7;15.3]. The mortality was significantly higher in the group of patients who obtained a specific diagnosis after transferral compared to the patients who maintained Z03diagnoses (P = 0.003).
Discussion Patients diagnosed with Z03* at hospital discharge have a substantially lower 30-day mortality, but a higher readmission-rate, compared to patients who obtain a more specific diagnosis during the entire hospitalization stay. Our findings corroborate and extend previous research on the use of unspecific Z03coding. The demographic characteristics of the patients in our study are in agreement with a national study by Vest-Hansen et al., while the median length of hospital stay was 3 days in our study compared to 1 day in their study . Our study also yielded a considerable lower proportion discharged with Z03diagnoses (8%) compared to close to 20% based on previous Danish studies [2, 5] . Vest-Hansen et al. included all medical departments in Denmark including cardiac departments , and two out of three patients were actually being observed for myocardial infarction (Z03.4, "Observation for suspected myocardial infarction"). In our study patients suffering from symptoms of myocardial infarction would be referred to a specialized cardiac department before admission to AMAU. Therefore, this would be a plausible explanation for the inconsistencies in results. In previous studies, the first admissions were primarily studied -building on this we followed the patients during the entire hospital stay, observing if the diagnosis changed. Overall more than half of the patients with Z03diagnoses at AMAU obtained a specific diagnosis before hospital discharge. The readmission rate was higher among the patients discharged with Z03diagnoses compared to the patients discharged after specification of diagnosis Fig. 3 30-day mortality following hospital discharge after initial Z03diagnosis, stratified on maintaining diagnosis or further specification during hospital stay. The readmission rates were generally higher than in previous studies in acute medical admission or emergency admission [3, 13, 14] . Although not directly comparable to Z-coding, the R-codes of the ICD-10 also describe non-causative diagnoses, and it is suggested that patients getting R-codes is "in less need of acute care and (admissions) may be avoidable if appropriate services are in place" . Could this also be true for the Z03diagnosed patients, not obtaining specific diagnoses later in their hospital stay? Further investigation in whether admittance is avoidable for the Z03patients is warranted, as this study is based on few patients. The 30-day mortality rate among discharged patients who had maintained their Z03diagnosis was lower compared to the overall non-stratified mortality rate of 6% reported by Schmidt et al. after acute admission to a medical admission unit , while the patients with a specification of diagnosis was higher. The patients with the highest risk of death were those who after transferral obtained specific diagnoses. As early initiation of relevant treatment could reduce mortality, this group warrants further investigation. Our study's strengths include population-based design and complete follow-up for mortality. Use of routinely recorded diagnoses, collected independently of the study, reduced the risk of information bias. Data from MidtEPJ is transferred to DNPR, which is known as a valid datasource for covering acute admissions to medical departments [15, 16] . This study only includes patients with admission at AMAU and readmission at any hospital department in Aarhus. If a patient was transferred or readmitted to a department outside Aarhus, we would count them as discharged to home, but we assume this potential misclassification to be of minor influence. Potential coding inaccuracy is an important limitation. The high and likely increasing proportion of imprecise Z-codes points to several potential healthcare problems. Firstly, it may suggest that some index conditions are improperly coded and underreported in the DNPR, which has direct impact on surveillance and research of important specific diseases. Second, frequent use of primary Z03diagnoses may hamper comparative hospital survival statistics such as hospital standardised mortality ratios where survival is compared for frequent primary diagnoses that account for a large proportion of deaths. Third, it may point to the presence of a growing vulnerable patient group with frequent contacts to hospitals with unspecific symptoms. Fourth, it raises the probability that diagnostic efforts are insufficient, and people with undetermined diagnosis may have insufficient therapy and increased risk of readmission and death. Our study adds to our baseline understanding of the characteristics and flow of the many patients with initial imprecise Z-codes through further departments and admissions, and quantifies their risks of readmissions and mortality. Future clinically detailed studies should investigate the exact reasons for potentially avoidable readmissions and adverse clinical outcomes in these patients.
Conclusion Patients diagnosed with Z03* at hospital discharge have a substantially lower 30-day mortality, but a higher readmission-rate, compared to patients who obtain a more specific diagnosis during the entire hospitalization stay. Fig. 1 1 Fig. 1 Length of stay
Table 1 1 Baseline characteristics of the 409 patients discharged from the AMAU with a Z03diagnosis Number Percent
|
10.3390/su13042305
|
cc-by
| null | null |
openalex
|
Designing solar strategies is a powerful step forward to set up an adequate residential house in terms of energy. Many types of research have simulated the energy needs for residential buildings. Designing an improper installation can contribute to a growth in the overall energy expenditure in ensuring thermal comfort. The use of solar thermal processes in Slovakia is on a rise as compared to recent years. This study models twelve solar water heating systems created on the roof of the household. Solar energy techniques are carried out to comply with the demands of heating and domestic hot water. The analysis deals with the most efficient alternative for the arranged solar systems of the building. Considering these installations and the corresponding overall prices of machinery, the best workable alternative is selected. The potential energy performance of auxiliary heating and the energy output of the solar thermal installation are examined. The required amounts of the different energy contributions are modelled and simulated in specific software for a family house in Kosice, Slovakia. We determine the limits of the design for an apartment and analyse which procedure is used to provide the typical average water expenditure and heating need, covering a multi-criteria analysis considering costs, energy, and life cycle analysis of every installation. This approach can support professionals to decide the best scheme considering these criteria, and this method can be satisfactorily applied. In these conditions, converting a conventional gas boiler into a solar thermal system involves monthly economic savings of around EUR 140-250, with payback periods of 2.5-7 years. The energy requirements are fully covered by the solar thermal schemes and the life cycle assessment resulted in reasonable impacts on the environment.Introduction The latest data stated that world energy production has continued to grow (+1.5%) in recent years and is close to reaching 15,000 Mtoe, but remains below its real trend (2%/year) . The energy demand is projected to rise by 1.3% each year until 2040 , and the population forecast highlights that the population will be 70% higher in the year 2050 . The overall energy expenditure in 2035 will amount to around 32,922 TWh, twice that in 2008 (with 75% of the overall energy used being produced by fossil fuels). The European Commission points out as one of its primary purposes curtailing emissions by 2050; such contraction can be achieved with monetary, industrial, and civil transformation measures . In 2018, the European Union (EU) fulfilled 55% of its energy demand using gas and oil sources, and encourages diminishing this value by up to 20% by the year 2050. The construction sector uses 40% of the overall energy consumption within the European Union. Energy management to reduce carbon emissions was revealed as a hot topic, as 12% of the literature produced is focused on this , and the concept of a postcarbon city appeared as a result of meeting the European targets regarding emissions. This industry handles a significant part of the overall greenhouse gas emissions. The European Union is trying to improve the environment in households by means of new ideas. One project is run by the Slovak Innovation Energetic Agency (SIEA), in which the state can support households (up to half of the cost) to perform any installations using renewable sources. The massive consumption of fossil fuels could likewise lead to important environmental concerns, such as air pollution and global warming . To promote energy self-sufficiency, the European Parliament has encouraged policies supporting energy efficiency in the construction industry. Regulations include energy certification of houses and economic motivations for reducing energy usage in residential buildings. Houses and the construction industry consume 33% of the overall final energy use and produce 40% of the total CO2 emissions . These figures justify the attempts to diminish energy use through envelopes [9, 10] , warming and cooling (60% of the overall consumption ), demand response, etc. In accordance with Directive 2010/31/EU, all EU Member States are obligated to perform energy analysis to diminish energy needs. The concept of net-zero energy buildings comes out as a solution for houses with an excellent energy performance and diminished energy need , where renewable sources feed energy demands. In this regulation, the cost-optimal method was proposed-this means dealing with the global costs applied to the building lifespan . Researchers and practitioners have investigated many techniques to exploit solar power for cooling and heating [15, 16] , photovoltaic energy for electricity , and hybrid solar collector thermal and photovoltaic systems . Many approaches have dealt with energy storage [19, 20] , with wind energy production [21, 22] , and with modelling energy demand in buildings [23, 24] . The energy retrofitting of existing buildings is also a challenge for diminishing energy consumption [25, 26] . Many studies have highlighted solar thermal (ST) installations as a promising and ecological technique [27, 28] , as they contributes to hot water management in several regions with changing solar irradiance . Despite systematic campaigns, the full-scale adoption of solar panels has not materialised yet, because of a lack of confidence in power savings [31, 32] . Solar energy usage is needed to face urgent dilemmas posed to our civilization, such as fossil fuel depletion, excessive CO2 emissions, and the increasing electricity demand . The life cycle analysis of solar technologies has been investigated , dealing with renovation materials in family houses [35, 36] , buildings [37, 38] , and commercial building . The studies found the most sensitive parameter is daily hot water consumption [34, 40] when designing a solar thermal system . Solar thermal energy generation can be used over a small scale or in the industrial sector . The researchers analysed the successful installation of solar concentrator systems [44, 45] because of the exorbitant amount of energy savings (40% ). Some approaches optimized solar thermal installations , considering the key components-the solar collector, the pump, and the storage tank. The auxiliary energy requirements were estimated and the tank for hot water storage was dimensioned to reduce its size . Solar power conversion systems need greater expenditures than conventional energy conversion apparatuses. However, they still provide future monetary savings (users will not pay for warming their houses and for obtaining domestic hot water (DHW)). The future costs or revenues (annual charges and/or future savings) incorporate a discount rate that accounts for inflation to obtain their present value. This work deals with assessing the overall cost throughout the system. First, for the best hypothesis of a lifetime period, it is compulsory to obtain the input data of energy needs for all installations. To follow these conditions, we simulate using the program "T*SOL" (Valentin Software) . The T*SOL software reports auxiliary heating energy requirements, the solar contribution to heating and for DWH for every alternative. Twelve ST schemes were analysed, and their net present value throughout their lifespan was calculated . These cases were selected as they are the most common schemes (I-IV) and incorporate different auxiliary heat sources. The energy savings were calculated by comparing each alternative with a zero-case (no renewable energies), covering a tank and a gas boiler for heating and DHW. We rank alternatives for the best alternative solar energy system in buildings after the machinery lifespan. In brief, higher revenues achieved after the installation lifespan indicate a better choice. In these cases, the climate conditions for Košice (Slovakia) were used. Without any doubt, choosing the appropriate solar thermal system in a cold climate condition represents a challenge due to the large amounts of energy needed and the irradiance limitation because of the latitude . A life cycle analysis (LCA) covering the whole installation (buffer tanks, ST panels, etc.) was performed for every alternative analysed. This research was carried out using software "OneClickLCA", attaining the impact on the environment in several categories. Bionova Ltd. developed OneClickLCA software compliant with EN 15978 standard [53, 54] . Afterwards, a multicriteria analysis (using MCA7 software) was performed based on proportion share for each of these categories. This analysis provides us with results for our decision. We set up the work as follows. Section 2.1 selects the input data required for the method, Section 2.2 shows the program, and we present the obtained output data in Section 2.3. Section 2.4 illustrates how to estimate the net present value, Section 2.5 shows the life cycle analysis of this installation, and Section 2.6 presents the multi-criteria analysis. Section 2.7 depicts the process. Section 3 shows a true case study. The results and the discussion are shown in Sections 4 and 5, while Section 6 features key conclusions.
Materials and Methods
Input Data for the Simulation The input data required for the procedure can be classified into building location, thermal installation, and economic data. Building location data cover longitude, latitude, diffuse radiation percentage, total annual global irradiation (kWh/m 2 ), average outside temperature (C), and the lowest outside temperature. The thermal installation data are gross collector area (m 2 ), active solar surface (m 2 ), active collector area (m 2 ), inclination (degrees), orientation, and azimuth angles (degrees). The economic data are the equipment to buy, the costs of the energy, and the discount rate.
T*SOL Software Characteristic T*SOL is a software for modelling ST installations in buildings. This program can reproduce solar thermal schemes for various households, comprising installation presentation of competent solar thermal systems and yield and assumption of profit. There are 80% of common solar installation variations in the database for such buildings in the USA and Europe. By implementing an alternative relation, the software detects the effect of solo pieces on the behaviour of a system. The variant study also presents the effects of global radiation, DHW, space heating, collector performance, auxiliary for heating, and total simulation outcomes. Software results show the scheme of behaviour in other outdoor conditions (the installation's location area, the value of heat loss of a building, etc.). Concerning the results achieved, a specific correlation between the variations and their costs was determined.
Output Data for the Annual Simulation T*SOL program calculates the following results for every case: Energy delivered by collectors (kWh). The software shows the energy produced by the solar thermal installation. It also divides this energy into solar energy increase to DHW (kWh) and heating (kWh).
Auxiliary heating energy (kWh). This value showed that our installation cannot offer the total energy demanded by our building. This energy can be achieved by different sources such as gas boilers, heat pumps, wood pellet boilers, and many others. In this work, the method is described, and practitioners can incorporate any other energy sources. The software highlights the wood pellet economic savings obtained by wood fire boilers (kg), natural gas savings by gas fire boilers (m 3 ), and the CO2 emissions avoided by heat pumps (kg).
Life Cycle Cost of ST Installation Maintenance may increase the lifespan of solar thermal systems (30 years). The ST modules need to be regularly cleaned. However, maintenance considers components such as thermostatic valves, pumps, solar circuit pumps, and storages for solar accumulation. This procedure intends to bring knowledge to professionals when they analyse which alternative technique is better. The upcoming incomes and savings in present-day money are considered here. Because the cost of capital is time-dependent, future costs and revenues must consider inflation to compare with today's money [55, 56] . The "Net present value" shows current investments and future earnings using the equivalent discount rate. This indicator must be maximised from the present time to the lifespan (Equation ( 1 )). We express this as the net present value (NPV) from the current year (0) to the installation lifespan (T*): NPV I e S e * dt (1) Ii is the investment performed, and Si is the monetary savings for the ith year. The maintenance costs should be incorporated into this analysis as a cumulative investment per year.
Life Cycle Assessment Utilizing the LCA assessment process, the environmental performance of a family house based on "cradle to the gate with options" is calculated. LCA is a methodical technique for determining the possible environmental effects associated with the lifespan of a product [57, 58] . The LCA method comprises all steps, which start from natural materials to industrial productions, and incorporates material extraction, energy expenditure, production, transport, usage, recycling, and ultimate removal or end-of-life. It is an overall process that specifies the influence a device can have on climate change, non-renewable resources, and the environment as an entity. As already mentioned, this LCA was performed using OneClickLCA software, which generates impact complying with standards of ISO 14040, ISO 14044, EN 15804+A1+A2, and ISO 21930 and ISO 14025. It complies with the CML-IA method. This software is a standardised program to run LCA with a good opportunity to cut down costs, incorporating environmental effects. The software OneClickLCA uses elements in management and construction stage through the use-step until end-of-life, i.e., the "grave" stage.
Multicriteria Analysis To determine which alternative is the best, according to environmental, economic, and energy aspects, we used the multi-criteria analysis (MCA). MCA is a family of wellknown methods implemented by decision support systems to compare different alternatives based on multiple factors, and to find the best performing solution . Due to various objectives considered in the analysis, the hierarchy approach was applied. For the calculation, the MCA7 software was used, which performs calculations using several specific methods with the so-called "Cardinal information on the criteria". This means that to use these methods, it is necessary to know (i) the criteria according to which the decision will be made (number of criteria, names of criteria, weights of criteria, and distinction between maximization and minimization criteria); and (ii) variants, or alternatives, between which decisions are made and which are arranged from best to worst using some multicriteria methods (number of variants, names of variants, values of all criteria for a given variant). For the multi-criteria analysis, MCA7 software was chosen, and the Concordance Discordance Analysis (CDA) method was used. CDA includes a comparison of alternatives for pair choice. It measures the degree by which the alternatives of choice and the weights of factors prove or disprove the ratio between the alternatives. The discrepancies in the weights of factors and the evaluations of criteria are analysed through the procedures of concordance and discordance , which the software calculates by itself from input data.
Calculation Process Figure 1 reflects the computation process in 10 stages. The irradiance inputs are gathered in step 1. The cases for solar thermal installation are depicted in step 2. The cases include supplementary heating provided by a gas boiler, a heat pump, and a wood pellet boiler. Calculations are performed using the software in Step 3 to obtain the energy results described before (Section 2.3). We consider the economic input data (step 4) and calculate the investment and savings for every case (Step 5). The economic analysis for every installation lifespan is calculated in step 6 to obtain the best alternative for this economic standpoint. The LCA is performed in step 7 using the parameters calculated in steps 2 and 3, obtaining the results shown in stage 8. Finally, results from stages 3, 6, and 8 are included in the MCA software to achieve a priority ranking in step 10.
Case Study
Input Data for the Simulation Table 1 shows the input climate and solar thermal installation data. Twelve opportunities to size solar thermal panels and other machinery were analysed (Table 2 ). The following step calculates the investment cost for every installation and every device of the installation. Another step of this analysis is to obtain midterm energy results from the software and compare them with measured values on a real house. For comparison, energy needs for an auxiliary source for heating were used, such as a gas boiler, heat pump, or wood boiler. After comparing the heating energy, it is also necessary to compare the domestic hot water (DHW) heating energy supply. CO2 emissions per year were also compared among alternatives. The last step calculates total lifespan for every situation. The designed one-floored apartment (usable area 53 m 2 ) is located in Košice (Slovakia). The heat load was 5 kW, the indoor temperature was 20 C, the specific head load was 94.34 W/m 3 , and the specific annual energy supply was equal to 163.585 W/m 3
Cases Simulation We describe the cases analysed here in Figure 2 . Case 0 does not have any solar thermal collector. It includes only a 15kW gas-fired boiler. Every case has five Thermosolar Žiar TS 300 solar collectors (with efficiency ηcol = 57%), and the components are described here. Case I has a 200 litre preheating tank, a space-heating buffer tank of 500 litres, one tank for DHW with 120 litres, and a gas-fired boiler with 15 kW performance and floor heating. Case II has a gas-fired boiler (15 kW), a 500 litre tank for floor heating, and a dual coil indirect water tank of 300 litres. Case III has a 1000 litre tank for DHW and floor heating and a gas-fired boiler with 15 kW performance. Case IV contains a gas-fired boiler with 15 kW performance and a 500 litre tank for floor heating and a heat exchanger. The following cases repeat the features in Cases I-IV but consider a heat pump (Cases V, VII, IX, and XI) or wood-fired boiler (Cases VI, VIII, X, and XII). To clarify this, the auxiliary source in Case I is a heat pump with 14 kW performance and floor heating (Case V), and this is compared to a wood-fired boiler with 14 kW performance and floor heating (Case VI).
Economic Data The financial character of input data collected from the T*SOL simulation software is summed up in the following paragraph: Table 2 shows all economic costs and their lifespans . Replacement costs are considered as investments in future time (they appear when the lifespan of every component ends). As replacement costs represent future expenses, their value is discounted by the equivalent continuous rate. Residual values of the replaced elements are considered as zero. As it can be observed in Table 2 , the best investment cost was case VI (4664.2 EUR), and the most expensive was case IX (20,828.89 EUR). The values for midterm energy calculations were determined from auxiliary heating, a gas boiler with 15 kW performance, heat pump 14 kW, and wood fire boiler with 14 kW. Gas boiler cases ranged from 7253 to 4981.2 EUR (cases III and I), heat pump cases ranged from 20,828.89 to 18,556.2 EUR (cases IX and V). Wood pellets boiler oscillated among 6936.89 and 4664.2 EUR (cases X and VI). Energy cost of natural gas (assuming 228 g per kWh ) for all alternatives was 1.32 EUR/kg. Other energy sources were wood pellet alternatives (0.18 EUR/kWh) and heat pump (0.08 EUR/kWh).
Results
The Energy Required for Every Alternative The energy required from this source according to the simulations in every case was 8900 kWh a year (Table 3 ). For this house, five solar panels (TS 300, Thermosolar Žiar) were installed to provide 120 litres of DHW per day.
The Energy Required for Heating Measured values from real-time were from the house in Košice with the same installation and were recalculated on the object area. Here, the highest potential value of simulation from the case was used. For instance, cases V, VII, IX, and XI included a heat pump as an auxiliary heat source. In the next case, VII had the highest value from these four cases (8970.8 kWh). The energy required for auxiliary heating-heat pump in real-time was 21.34 kWh/ (m 2 a year). We calculated this value by the object area (53 m 2 ), and the result was 3467.79 kWh a year. Results are depicted in Table 4 , highlighting differences between software and real-time values.
Domestic Hot Water Table 4 presents the DHW demand. The energy production from an auxiliary source in each case was due to the equal energy from the solar thermal panel and their amount in every installation. The values from the simulation software of energy for DHW were the same for each case (2040.65 kWh). The energy demand for DHW with a heat pump as another heat source was 14.5 kWh/ (m 2 a year) in real-time. We counted this value by the object area (53 m 2 ), and the result was 768.5 kWh a year. As it can be observed, the values from real-time were in the interval of 725-792 kWh. The lowest value for DHW energy supply was for the solar thermal installation with a gas boiler, and the best was from a wood-fired boiler installation.
Emissions and Impact on the Environment We obtained results from simulation software for CO2 emissions avoided and electricity savings for each of four alternatives, where a heat pump was an auxiliary heat source. In the wood-fired and the gas boiler cases, wood pellets and gas savings were calculated (Table 5 ).
Economic Savings of the Alternatives The economic energy savings with the data shown in Table 6 are calculated. The cost of the gas was calculated in the zero case. Every option produced an economic reduction. The costs of the pellets, gas, and more energy were considered for every choice.
Life Cycle Cost of the Solar Thermal Installations With the investments and lifespan shown in Table 2 and the savings depicted in Table 6 , the net present value was calculated for every case. To clarify the analysis, we showed the case I numbers. Case I contains five Thermosolar Žiar TS 300 solar collectors (2128.8 EUR, 30 years), a 200 litre preheating tank (401.4 EUR, 20 years), one tank for DHW with 120 litres (376 EUR, 15 years), a space-heating buffer tank of 500 litres (906 EUR, 15 years), and a gasfired boiler (1169.0 EUR, 15 years). Thus, this installation required buying some equipment (those whose lifespan is lower than 30 years) twice. The investment was calculated as follows: I 0 4981.2 376.0 1169 376 e 15r 401.4 e 20r (2) The economic savings were 159.4 EUR/month (Table 6 ), and the yearly savings resulted as 1909.71 EUR/year. With these numbers, we calculated the net present value: NPV ∑ I The evolution in the cost of the gas boiler cases is shown in Figure 3 . Table 7 shows the net present value for every solar thermal installation, and values were obtained by performing analogous calculations for every alternative. The effect of future replacement costs can be observed as decreases in the NPVs in Figure 3 (years 15 and 25).
Influence of the Discount Rate We consider the discount rate as a sensitive criterion. The present value of each cash inflow/outflow considers a discount rate with this parameter. The equivalent continuous discount rate is not modifiable by users as it depends on the national country banks (-1.3% in the UK, 2.2% in the USA). This influence is illustrated in Figure 4 , where high values of the equivalent discount rate reduce the economic differences for the installation scheme.
Life Cycle Assessment Calculation Table 8 presents output data from "OneClickLCA" software for all alternatives. This analysis provides quantitative amounts of the environmental impact groups such as global warming potential (GWP), ozone depletion potential (ODP), acidification potential (AP), eutrophication potential (EP), photochemical ozone creation potential (POCP), and non-hazard waste (NHW). They are expressed as kilograms of CO2eq, SO2eq, (PO4) 3-eq, CFC11eq, C2H4eq and NHW. According to the results, case IX had the biggest impact on GWP, AP, and ODP categories, while the highest values in EP, POCP, and NHW were found for cases XI, X, and XII. Case I had the lowest impact on GWP, AP, EP, ODP, and POCP. The lowest value in the NHW category was for case II. Percent representations of life cycle phases on environmental impact categories are depicted in Figure 5 . Modules A1-A3 represent the product phase (A1-raw material, A2-transport, A3-manufacturing), and module A4 represents transport to site. Modules B4-B5 represent the use phase (B4-repair, B5-refurbishment), and B6 represents operational energy use. Modules C1-C4 represent the end-oflife phase (C1-deconstruction, C2-transport, C3-waste processing, C4-disposal). Module D is benefits and loads beyond the system boundaries. We can see that the best environmental impacts were in the use phase, modules B4repair, B5-refurbishment, and B6-operational energy. Modules B4-B5 represent percentage of 20.8-47.9% for GWP; 13.8-44.7% for AP; 30.5-58.4 for EP; 19.2-53.6% for ODP; 25.4-54.0% for POCP; and 37.3-65.0% for NHW. Module B6 represents share of 21.8-48.1% for GWP; 28.1-60.5% for AP; 18.5-44.0% for EP; 28.6-71.1% for ODP; 18.4-39.3% for POCP; and 2.6-16.2%.for NHW. Module D, representing benefits (which have negative value), had a significant impact on the decrease in the total values of environmental impact categories, ranging 14.3-26.2% for GWP; 12.3-23.8% for AP; 3.6-15.4% for EP; 0.0-2.8% for ODP; 9.6-26.8% for POCP; and 10.8-35.1% for NHW. It also has to be mentioned that the product phase had impacts in the range of 4.3-11.9% for GWP; 2.6-10.8% for AP; 5.7-16.8% for EP; 3.7-11.3% for ODP; 6.1-17.1% for POCP; and 8.3-20.2% for NHW. Within "cradle to gate" (from resource extraction to the factory gate) effects (A1-A3), the most contributing components were solar thermal collector (30.5-74.% for alternatives 1-12); solar heat exchanger system (38.4-48.2% for cases IV, XI, and XII), heat pump (21.5-38.2 % for cases V, VII, IX, and XI), and wood fired boiler (31.1-50.4% for cases VI, VIII, X, and XII).
Multi-Criteria Decision Analysis To choose the best alternative in calls of environmental, economic, and energy conditions, a multi-criteria analysis was performed. Based on the CDA, the summary weight of all criteria needs to have a value of 1. We assigned 0.34 for environmental (0.102 for GWP and ODP; 0.034 for AP, EP, POCP, and NHW), 0.33 for economic, and 0.33 for energy criteria. Table 9 shows the results of the multi-criteria analysis; case VI was the best and case II was the worst.
Discussion The energy obtained by solar thermal modules was similar for every choice and, likewise, so was the supplementary energy needed by any other source (8900 kWh per year, Table 3 ). The results highlight that every solar thermal system is workable. In these conditions, we identified gas boiler alternatives (despite being economically viable) are far from heat pump cases. The wood-pellet boiler alternatives are the best economic options, as they do not represent a great expenditure and offer significant savings. The wood pellet boiler cases resulted in the least CO2 emissions, as depicted in Figure 4 . Among them, case XII appears to be the best economic choice (a wood fire boiler). Case XII is the best choice from the economic standpoint, but results are affected by the equivalent continuous discount rate. Small values of the equivalent discount rate mean greater revenues. To summarise, profits must be re-established to achieve a new apparatus as the upcoming savings will be discounted, and we obtain the pay off in a smaller stage. Practitioners consider the payback period (the time at which the full expenditure reaches the full energy cost savings on the buy) for a ST system. However, this approach may lead to avoiding economic savings for the system lifespan. The payback period analysis formulation can be calculated [55, 64] : T 1 r . ln 1 r. I S (4) We can get the values displayed in Table 10 . These results show that the strongest economical alternative would be case VI, not case XII. The payback period is not the proper index if only economic aspects are considered. The lowest payback period (case VI) gives lower benefits than case XII (Table 6 and Figure 4 ) for the life cycle cost analysis. So, we must select case XII from this economic standpoint. Based on the results of LCA analysis considering only environmental criteria, case IX using a heat pump is the best. Most contributing components for GWP of this alternative are a solar collector (61.7%) and heat pump (38.2%). In contrast, the worst is case I, which also has a solar preheating tank, domestic hot water standby tank, space-heating buffer tank, and gas boiler. Most contributing components for GWP of this alternative are a solar collector (67.4%), gas boiler (23.5%), and hot water tank (9.1 %). The total values of the environmental impact categories are presented in Table 8 . According to LCA analysis considering environmental, economic, and energy criteria, the best one is alternative 6. It also comprises a solar preheating tank, domestic hot water standby tank, space-heating buffer tank, and wood-fired boiler. Most contributing components for GWP of this alternative 6 are wood boiler (47.3%), solar collector (46.4%), and hot water tank (6.3%). Based on this study, considering all three aspects of sustainability, we can conclude the best is case VI. The study dealing with the LCA of solar thermal systems also compares flat plate and vacuum tube systems. This emphasizes that both collectors can encompass more than half of the yearly hot water requirements for a family house with four inhabitants. The solar fraction values for the vacuum tube and the flat plate collector are 62.7% and 55.3%. According to this approach , the domestic solar water heating system market advances to expand, but the success rate depends on many circumstances that are not controlled by users (the costs of solar collectors versus ordinary heating processes, government subsidies, and the energy prices). Besides, another study shows that the lowest increase in the technical building material on the overall LCA results was in a passive house because of the absence of "traditional" technical building machinery for heating. Figure 6 shows the best-evaluated case of solar thermal installations for different evaluation methods. It can be said that the evaluation of just one aspect may not indicate the best alternative. A comprehensive assessment of several aspects, especially energy, economic, and environmental, helps us find the optimal solution.
Conclusions The energy demanded by a residential building can be calculated using software, and the result shows higher demands than raw data from real-time. This need influences the solar thermal installation proposal for every case. Converting conventional gas boiler installations into ST systems may contribute to significant savings. However, this research also emphasizes that not dealing with the opportunities (wood pellets, boilers, or heat pumps) can offer considerable savings that are not handled. Wood-fired cases (XII, VIII, VI, X) resulted as the most interesting options only considering the economic aspects with a positive NPV oscillating among 54,261.96 and 61,262.56 EUR. All cases proved to be a good opportunity from an economic standpoint. This means it is feasible to convert gas boilers into ST systems. The cost analysis is proposed for assessing various options dealing with all the present expenditures and future earnings expressed in financial units at present (adopting the discount rate, r). This cost analysis reports the strongest opportunity, as with the highest savings for the installation lifespan. We propose a method to determine which alternative is more workable, bearing in mind auxiliary heating sources. The different energy contribution is considered in specific software. By dealing with future expenses and savings for the lifespan of the machinery, the best alternative among the options in a real family house is calculated We associate the greatest annual operating costs by preparing heat for heating and DHW. This study points out interesting information resulting from a comparison of heating systems and heat sources over 15 years, which correlates with the results of our study. It states that, regarding the total cost of the heating system during the 15-year evaluation period, wood heating is the most helpful, followed by natural gas, pellets, and a heat pump. If user comfort is included in the evaluation, then heating by natural gas is the most suitable choice in terms of price . The disadvantage is that solar energy was not applied to the heat source, which would bring the results of the analysis closer to our results. Regarding the energy requirements of every case, we can assure that every scheme compared achieves the demands, and raw data from real-time present lower values than those simulated (Table 4 ). Finally, the life cycle analysis highlights the environmental concerns for the entire cradle-to-grave process. In Slovakia, practitioners consider investment costs in the planning step of a building and technical systems. However, the costs of maintenance, system installation, and operation also describe a certain item in costs. Assessment of the life cycle costs can offer an optimal design and cost-effective heating and DHW system. Current global requirements to carry out a low-carbon environment force us to deal with environmental needs to a greater extent. Figure 1 . 1 Figure 1. Workflow for the process to calculate the lifespan of the alternatives.
Figure 2 . 2 Figure 2. Numbers represent the following: 1-solar collector Thermosolar Žiar TS 300; 2-solar preheating tank, 200 litres; 3-DHW (domestic hot water) standby tank, 120 litres; 4-gas boiler, 15 kW; 5-combination tank, 1000 litres; 6-dual coil indirect water tank, 300 litres; 7-space-heating buffer tank, 500 litres; 8-heat exchanger; 9-floor heating; 10-DHW consumption; 11-heat pump, 14 kW; 12-wood fired-boiler 14 kW.
Five solar collectors = 2128.8 € (this cost applies to all cases), type Thermosolar Žiar TS 300 flat plate collector, gross surface 2.03 m 2 , active solar surface 1.78 m 2 , gross collector area 10.15 m 2 , active collector area 8.9 m 2 , orientation 180, inclination 45, and azimuth angles 0; Gas boiler = 1169 € (cases I, II, III, and IV), nominal output 15 kW; Solar preheating tank 200 litres = 401.41 € (cases I, V, and VI), volume 200 litres, height 1.8 m, insulation thickness 100 mm, effective thermal conductivity 0.065 W/(mK), and losses 2.31 kWh/day;
Figure 4 . 4 Figure 4. Net present value in year 30 for the cases studied.
Figure 5 . 5 Figure 5. Percent representation of life cycle phases in environmental impact categories.
Figure 6 . 6 Figure 6. A diagram showing the best alternative in each evaluation method.
Table 1 . 1 Input climate data for a house and solar thermal installation. Building Location Thermal Installation Location Košice, Slovakia Latitude 48.7 Active solar surface 1.78 m 2 Longitude -21.3 Gross collector area 2.03 m 2 Overall global irradiation (tilt angle 45) 1144.4 kWh/m 2 Orientation 180 C Diffuse radiation 53.90% Lowest outside tem-perature -13 C Average outside tempera-ture 9.8 C Azimuth angles 0
Buffer tank 500 litres = 906 € (cases I, II, V, VI, VII, and VIII), volume 500 litres, height 2.93 m, insulation thickness 45 mm, effective thermal conductivity 0.03 W/(mK), and losses 3.46 kWh/day; DHW standby tank 120 litres = 376 € (cases I, V, and VI), volume 120 litres, height 1.8 m, insulation thickness 100 mm, effective thermal conductivity 0.065 W/(mK), and losses 1.95 kWh/day; Dual coil indirect water tank 300 litres = 1944.76 € (cases II, IV, VII, VIII, XI, and XII), volume 300 litres, height 1.8 m, insulation thickness 100 mm, effective thermal con- ductivity 0.065 W/(mK), and losses 3.23 kWh/day; Collector loop heat exchanger = 100 € (cases IV, XI, and XII), maximum heat transfer rate 4.45 kW; Combination tank = 3956.09 € (cases III, IX, and X), volume 1000 litres, height 2 m, insulation thickness 100 mm, effective thermal conductivity 0.065 W/(mK), and losses 4.75 kWh/day; Heat pump = 14,744.0 € (cases V, VII, IX, and XI), nominal output 14 kW; Woodfire boiler = 852.0 € (cases VI, VIII, X, and XII), nominal output 14 kW.
Table 2 . 2 Investment cost of equipment for cases I-IV. Equipment Case I Case II Case III Case IV Lifespan Solar collector TS 300 5x 2128.8 2128.8 2128.8 2128.8 30 Solar Preheating tank 200 li-tres 401.4 - - - 20 DHW standby tank 120 litres 376.0 - - 15 Gas Boiler 15 kW 1169.0 1169.0 1169.0 1169.0 15 Combination tank - - 3956.1 - 15 Dual coil indirect water tank 300 litres - 1944.8 - 1944.8 25 Space-heating buffer tank 500 litres 906.0 906.0 - 906.0 15 Collector loop heat exchanger - - 100.0 30 Total Investment 4981.2 6148.6 7253.9 6248.6 Equipment Case V Case VI Case VII Case VIII Lifespan Solar collector TS 300 5x 2128.8 2128.8 2128.8 2128.8 30 Solar Preheating tank 200 li-tres 401.4 401.4 - - 20 DHW standby tank 120 litres 376.0 376.0 - - 15 Heat pump-14 kW 14,744.0 - 14,744.0 - 15
Table 3 . 3 The energy required (kWh/day) from auxiliary heating. Gas Boiler Heat Pump Wood Pellet Boiler Case I 8970.7 Case V 8969.9 Case VI 8970.8 Case II 8911.9 Case VII 8970.8 Case VIII 8910.2 Case III 8934.1 Case IX 8931.5 Case X 8930.2 Case IV 8947.5 Case XI 8942.2 Case XII 8943.7
Table 4 . 4 Energy demanded by heating and DHW regarding simulation software and measured actual values (kWh/year). Heating Domestic Hot Water Cases Software Real-Time Software Real-Time ST heat pump 8969.90 1131.02 2040.65 768.50 ST wood-fired boiler 8943.70 4547.40 2040.65 725.57 ST gas boiler 8970.70 3467.79 2040.65 792.35
Table 5 . 5 CO2 emissions avoided for every alternative. Heat Pump CO2 [kg] El. (kWh) Wood-Fired Pellets [kg] CO2 [kg] Gas Boiler CO2 [kg] N.Gas [kg] Case V 254.06 381.5 Case VI 579.7 179.7 Case I 711.29 336.4 Case VII 247.24 371.24 Case VIII 563.6 174.7 Case II 684.79 323.8 Case IX 324.78 487.7 Case X 734.3 227.6 Case III 853.08 403.4 Case XI 246.29 369.8 Case XII 560.8 173.8 Case IV 681.55 322.3
Table 6 . 6 Economic monthly savings for every alternative. Case I Case II Case III Case IV 159.14 159.81 159.56 159.41 Case V Case VI Case VII Case VIII 247.47 230.73 247.56 230.94 Case IX Case X Case XI Case XII 247.53 230.87 230.87 247.51
Table 8 . 8 Output data from LCA software. Case GWP kg CO2eq AP kg SO2eq EP kg (PO4) 3-eq ODP kg CFC11eq POCP kg C2H4eq NHW kg I 10 549.77 48.39 17.83 0.00253 3.68 1 876.17 II 14 339.08 63.34 19.27 0.0033 3.80 1 247.27 III 24 869.33 109.32 29.09 0.0052 5.29 1 284.77 IV 21 190.66 92.92 34.97 0.0037 4.67 5 282.93 V 17 592.63 89.27 22.2 0.00398 5.33 1 833.69 VI 14 611.98 74.39 21.11 0.0029 9.11 6 178.25 VII 21 180.22 103.07 23.47 0.00468 5.39 1 197.44 VIII 18 401.30 89.34 22.55 0.0036 9.23 5 549.34 IX 34 000.20 162.11 35.26 0.00698 7.51 1 318.23 X 28 931.55 135.32 32.37 0.0056 10.72 5 586.85
Table 9 . 9 Results of the CDA method from MCA7 software. Ranking Priority Score Ranking Priority Score 1 Case VI 4.4814 7 Case XI 10.3488 2 Case IX 5.3568 8 Case I 11.2578 3 Case XII 5.5442 9 Case IV 12.1477
Table 10 . 10 Payback period (years) for every alternative. Case I Case II Case III Case IV 2.68 3.31 3.94 3.38 Case V Case VI Case VII Case VIII 6.68 1.71 6.77 2.15 Case IX Case X Case XI Case XII 7.56 2.57 7.16 2.04
|
10.1175/bams-d-23-0216.1
|
cc-by
| null | null |
openalex
|
I n his comment (Kowalski 2023 ) on our recent publication (Heiskanen et al. 2022) where we present the Integrated Carbon Observation System (ICOS) research infrastructure, Andrew Kowalski introduces three important and, in our opinion, different potential issues in the definition, collection, and availability of field measurements made by the ICOS network, and he proposes possible solutions to these issues.
It is a density The first comment by Kowalski is about the definition of the "flux density" that we, in ICOS and in general in the FLUXNET community, simplify and shorten as "flux." There is no doubt that what Andrew Kowalski nicely explained with a clear example using radiation to illustrate the difference between flux and flux density is absolutely correct. If on one side the measurement units (W m -2 or μmolCO 2 m -2 s -1 ) clearly define that nature of the variable reported (a flux density), it is also correct to point out that the right definition should be used, at least in the description of the variables. This will probably not avoid that the commonly used short name "flux" will continue to be used to designate the flux density measured at the eddy covariance stations. However, the use of a correct naming in the official documents and portal will at least help to clarify the correct definition. For this reason we support the made by Andrew Kowalski to update the flux density entry in the Glossary of Meteorology (American Meteorological Society 2022), while for the flux definition, where we agree on the proposed text, we also suggest to keep the second point in the glossary, namely, "In the field of atmospheric turbulence and boundary layers, often used as a contraction for flux density, namely, the flow of a quantity per unit area per unit time." The abbreviated term was and will be largely used, and it is important that the AMS Glossary reports this information. At the same time, we also agree that in the ICOS Carbon Portal the term "flux density" should be used at least in the official variables' definitions, and the ICOS Ecosystem Thematic Centre (ETC) will ensure that the correct terminology is used. Where are the data? The second comment and the corresponding suggestion are about the recording and availability of the turbulent flux densities along the x and y directions (horizontal with respect to the rotated sonic anemometer wind vectors), arguing that these can be still relevant in the fluxes' computation and interesting for scientists, given the fact that the eddy covariance technique is still not definitive and can still evolve. It is first important to clarify that the eddy covariance stations record high-frequency data of the three wind vector components, sonic temperature, and scalar concentrations. For this reason, the turbulent flux densities in the three directions can be always calculated from the original measurements, and so stating that these data are not recorded is not fully correct. On the data availability, it is important to remark that ICOS is a fully open access Research Infrastructure, where all data (from raw data to final products) and all codes used to generate the products are available to all users, under a CC BY data policy, and that this is a pillar of the ICOS philosophy. We calculate and derive, solely for the eddy covariance measurements, more than 130 output variables and products that are distributed by the ICOS Carbon Portal. There will always be variables that could be potentially interesting and that are missing from this list, but this is the unavoidable compromise between providing useful information and keeping the whole system manageable, ensuring the maximum quality. A variable, when provided by ICOS, must be quality controlled, with full traceability and respecting the Findable, Accessible, Interoperable, Reusable (FAIR) principles. The selection of variables to be routinely provided has been defined on the basis of what the user community generally requests and searches. The ICOS ETC is, however, always offering to provide on request the hundreds of "secondary variables" that are produced during the standard data processing.
Parallel to what? The third aspect covered by Andrew S. Kowalski in his comment is related to the orientation of the radiation sensor in the eddy covariance sites. He states that "if the PAR sensor and ecosystem are not parallel, then the measured flux systematically misrepresents the ecosystem flux," and for this reason he suggests that for "fluxes measured by single-surface radiation sensors [] such sensors should be oriented with care to ensure that the measured flux corresponds to the flux of interest." In this case, however, the question would be to define and measure which is the right orientation. Landscape, when not perfectly horizontal, like in the case of some agricultural fields or lakes, is rarely with a homogeneous and constant slope and aspect. In addition, one could argue that the radiation flux density relevant for the ecological processes is the one happening toward the leaves, that could have a predominant orientation, not always (or better, rarely) parallel to the orographic slope. In addition, even hypothesizing that a representative nonhorizontal orientation could be unequivocally and standardly defined, the precise installation of the radiometers with the orientation parallel to this surface would be practically impossible or prone to rather important errors. The sensors are often a few centimeters in diameter are mounted over high towers, where ensuring the horizontal position using the bubble spirit level is hard and requires periodic fine adjustments. For this reason, we think that the correct way to measure these fluxes is to follow a common standard and then consider the specific elements structure (orography, vegetation, leaves, etc.) when the measurements are analyzed and interpreted. In fact, this is also the standard followed by the WMO that, in its Guide to Instruments and Methods of Observation (WMO 2021), suggests installing the pyranometers "levelled [] so that, when properly exposed, the receiving surface is horizontal, as indicated by the spirit-level." In ICOS, from the first definition of the protocols and procedures, it was decided to follow, whenever available, internationally recognized standards in order to maximize the level of interoperability. This is why the WMO and ISO standards on meteorological variables are the basis of the ICOS protocols. For this reason, although we agree that the geometry of the flux-surface interaction and, in particular, the incidence angle should be always considered, we think that this should be done after the measurements collection, which instead should follow a clear, unequivocal, and practically feasible-to-apply standard setup protocol.
|
10.3897/phytokeys.210.90598
|
cc-by
| null | null |
openalex
|
A new species of Smithia Aiton, S. yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng, sp. nov. from the wetlands of Taiwan is reported in this article. This species was mistakenly identified as S. sensitiva Aiton, but can be distinguished by its pale yellow corolla (vs. vivid yellow), often smaller flowers and shorter style. There is also a color gradient on the adaxial surface of the leaflets between young and mature leaves. Surface sculpture of pollen of S. yehii has significantly larger perforations, and muri are wider than those of S. sensitiva. An identification key to the Smithia taxa of Taiwan and S. sensitiva is presented.Introduction The genus Smithia Aiton belongs to the tribe Aeschynomeneae (Benth.) Hutch., Papilionoideae DC., Leguminosae Juss. (LPWG 2017) and contains c. 20 species (Sa and Delgado-Salinas 2010) . The genus is widely distributed in the tropics, chiefly in Asia and Madagascar (Huang and Huang 1987; Klitgaard and Lavin 2005) . There are fourteen species in India (Balan and Predeep 2017) , five species in China (Sa and Delgado-Salinas 2010) , and two species in Taiwan (Huang and Ohashi 1977; Huang and Huang 1987, 1993) . The first record of Taiwanese Smithia was made by Forbes and Hemsley (1887) , in which they recorded the species, S. sensitiva Aiton. Next, Hayata (1911) described a new species, S. nagasawai Hayata, based on its truncated or round apex of bracts, which differ from the acute apex of bracts in the similar S. ciliata Royle. Later, Hosokawa (1936) recorded S. ciliata in central Taiwan. Huang and Ohashi (1977) treated S. nagasawai as a synonym of S. ciliata. Since then, all authors have treated only two species of Smithia in Taiwan in subsequent papers (Huang and Ohashi 1977; Huang and Huang 1987, 1993) : S. sensitiva and S. ciliata. During a recent field and herbarium investigation, we noticed that the identity of S. sensitiva was somewhat controversial in Taiwan. Specimens initially identified in the field as Smithia sensitiva had vivid yellow flowers up to 1.5 cm long with styles up to 8 mm long (Aiton 1789; Efloraofindia 2007 onwards; Sa and Delgado-Salinas 2010; Balan and Predeep 2017) . However, all specimens previously identified as S. sensitiva in Taiwan had smaller pale yellow flowers with shorter styles. Hence, it was suspected the Taiwanese population was likely an unknown taxon distinct from S. sensitiva. The aim of the present study was to elucidate the taxonomic status of this taxon by morphological and palynological approaches.
Materials and methods
Morphological comparison We compared three Smithia taxa including Taiwanese taxa, viz. S. ciliata and the unknown taxon, together with its similar species, viz. S. sensitiva, which were collected from herbaria (see additional specimens examined). Morphological measurements were made on both fresh and dried materials. For the morphological description, the terminology followed the studies of Sa and Delgado-Salinas (2010) and Balan and Predeep (2017) .
Herbarium resources Herbarium acronyms followed Index Herbariorum (Thiers 2022, continuously updated) . Voucher specimens collected for the current study were deposited in TCF and TNM. Physical or digital specimens from the following herbaria were examined: HAST, PH, TAI, TAIE, TAIF, TCF and TNM. Type information of S. sensitiva followed the study of Balan and Predeep (2017) .
Pollen morphology We compared the pollen morphology of the unknown taxon with that of its similar species, S. sensitiva, and information about the voucher specimens is provided in Table 1 . Pollen materials were treated according to the methods of Schols et al. (2004) and Halbritter (1998) . Pollen grains were obtained from herbarium materials and isolated anthers were rehydrated overnight. Whole anthers were fixed in 2% glutaraldehyde overnight, then treated with DMP (2, 2-dimethoxypropane) for 30 minutes, and transferred to acetone for 30 minutes before critical-point drying (CPD). Dried pollen was mounted on a stub and sputter coated with gold for > 100 s (Quorum SC7620) and examined by scanning electron microscopy (Hitachi S-3400N). The terminology for pollen shape, size, and exine ornamentation followed the recommendations of Erdtman (1952) and Halbritter et al. (2018) . The quantitative palynological traits were measured and their means and standard deviations were calculated. For each quantitative character, the Shapiro-Wilks normality test was first used to check the distribution, then an independent sample t-test was performed after logarithmic transformation (Kim 2015) . All analyses were done using the PASW Statistics ver. 18 software (Sarma and Vardhan 2018) .
Distribution map The occurrence data was based on herbarium specimens. A distribution map was generated by using the package of Lin (2018) for QGIS ver. 3.4.
Results and discussion We compared the macro-morphology of the three Smithia taxa, S. sensitiva, S. ciliata and S. yehii (Fig. 1, Table 2 ) and the pollen morphology between S. sensitiva and S. yehii (Fig. 2 , Table 3 ).
Macro-morphological differences Smithia ciliata is distinctly different from other species in that its inflorescences often have more than twelve flowers (Fig. 1C ), whereas those of S. sensitiva and S. yehii have fewer than seven flowers (Fig. 1 A, B ). The calyx of S. ciliata is densely ciliate at the margin, and membranous with clearly reticulate veins, while S. sensitiva and S. yehii have entire margins and scarious parallel veins. The pods of S. ciliata are slightly orbicular and often more Huidong collector team 730 (TNM) than 1.1 cm long, whereas both S. sensitiva and S. yehii are more or less straight and usually less than 1 cm long (Huang and Huang 1993 ; Sa and Delgado-Salinas 2010) (Table 2 ). Compared with S. sensitiva, the corolla of S. yehii is pale yellow (Fig. 1A ); whereas S. sensitiva has a vivid yellow corolla (Fig. 1B ). S. yehii often has smaller flowers (0.7-1.0 cm long) than S. sensitiva (0.8-1.5 cm long). In addition, S. yehii has a shorter style 2 ). Leaves of S. yehii are usually smaller (3.5-7.0 mm long) with fewer than nine pairs of leaflets, while S. sensitiva often has up to eleven pairs of leaflets and they are larger (up to 1.5 cm) (Efloraofindia 2007 ; Sa and Delgado-Salinas 2010) (Table 2 ). Furthermore, S. yehii has color variations on parts of the adaxial surface of the leaflets, with dark green at the apex and light green at the base (Figs 1A, 3B, F); older leaflets are consistently dark green. S. sensitiva leaflets remain consistently pale green (Fig. 1B ).
Pollen morphological differences The pollen grains of both S. yehii and S. sensitiva are small, tricolporate, and spheroidal with perforated exine ornamentation. Smithia yehii has significantly larger exine perforations (0.2-0.6 μm) than S. sensitiva (0.1-0.3 μm) (p = 0.000*), and S. yehii has significantly larger muri (width of 0.3-1.1 μm) than S. sensitiva (0.3-0.7 μm) (p = 0.044*) (Fig. 2 , Tables 3, 4 ). The pollen characteristics also support the two taxa as distinct species. S. sensitiva sensu acut. Forbes and Hemsley, J. Linn. Soc., Bot. 23: 170, 1887; Henry, List 32, 1896; Matsumura, Bot. Mag. (Tokyo) 16: 73, 1902; Hayata, Icon. Pl. Formosan. 1: 180, 1911; Hosokawa in Masamune, Short. Fl. Formosa 106, 1936; Chuang and Huang, Leg. Taiwan Past. 93, 1965; Huang and Ohashi in Li, Fl. Taiwan 3: 381, 1977; Huang and Huang, Taiwania 32 (1): 88, 1987; Huang and Huang, Flora of Taiwan, 2 nd edition 3: 364, 1993, non Aiton.
Diagnosis. The new species is similar to S. sensitiva, but can be distinguished by its pale yellow corolla (vs. vivid yellow), often smaller flower and shorter style, and color variation on adaxial surface of leaflets when young and mature, viz. dark green at apex and light green at base. Type. Taiwan. Miaoli County: Tunghsiao Township, Tunghsiao Township 14 th Cemetery, 81 m alt., 24.44718N, 120.69563E, 17 Dec 2021, C.M.Wang 19231 (holotype: TNM) (Fig. 6 ). Description. Diffuse annual herb, 25-50 cm long; stem slender, sparsely bristly. Stipules 2.7-5.5 × 1.0-1.6 mm, ovate, striate, scarious, persistent; appendage to the stipules 1.9-3.6 mm long, bilobed. Leaf rachis bristly; petioles 0.9-1.6 mm long; leaflets (2)4-9 pairs, 3.5-7.0 × 1.2-2.3 mm, linear-oblong, obtuse at apex, mucronate, oblique and truncate at base, bristly beneath along the midvein and margins; adaxial surface dark green at apex, light green at base; older leaflets consistently dark green. Racemes axillary, 1.1-3.4 cm long, 1-7-flowered; peduncles filiform, sparsely bristly. Flowers 0.7-1.0 cm long; pedicels 1.0-3.1 mm long; bracteoles 2.3-4.0 × 0.9-2.4 mm, ovate, striate, persistent. Calyx parallel-veined, lips 4.5-8.2 mm long, equal, ovate, acute at apex, with a few scattered bristles. Corolla pale yellow, standard (5.2)6.2-9.0 × 5.7-8.0 mm, obovate, pale yellow with red circle pattern in centre; wings 4.0-6.9 × 1.8-2.6 mm, oblong, auricled; keels 4.8-7.5 × 1.9-2.5 mm, oblanceolate. Stamens diadelphous; filaments 5.1-6.4 mm long; anthers 0.2-0.3 mm long, ovoid. Ovary Chinese name. yè-shìh-po-yóu-gan (葉氏坡油甘). Etymology. The species epithet "yehii" was chosen to honor Prof. Mau-Shing Yeh (葉茂生), Department of Agronomy, National Chung-Hsing University, for his contributions to research into the legumes of Taiwan. Palynology. Pollen grains are small, tricolporate, and spheroidal, perforate in surface sculpture, and 19.6-22.7 × 16.5-21.9 μm, P/E ratio 0.9-1.3, perforations 0.2-0.6 μm in diam., and murus width 0.3-1.1 μm (Fig. 2A-D ). Conservation status. Smithia yehii was evaluated as least concern (LC) by the Editorial Committee of the Red List of Taiwan Plants (2017) as S. sensitiva, because there were many records in the herbarium. However, many populations are probably extinct now. Smithia yehii is known after 1950 from only four sites (Fig. 5 ), each of which had only a few individuals (c. < 30) because of human disturbances and habitat fragmentation. Therefore, following the criteria of IUCN (2019), we regard this species as endangered (EN B2ab(ii, iii) ; C2a(i); D), and recommend that it urgently needs to be protected against extinction. Figure 1 . 1 Figure 1. Comparison of Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng and its similar species. Scale bars: 3 cm A S. yehii (photo by C.M.Wang, from Miaoli, Taiwan) B S. sensitiva Aiton (photo by Chih Y.Chang, from Chiang Mai, Thailand) C S. ciliata Royle (photo by C.M.Wang, from Chiayi, Taiwan).
Figure 2 . 2 Figure 2. Comparison of the pollen morphology of Smithia Aiton A-D Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng E-H S. sensitiva Aiton A, E polar view B, F exine ornamentation of polar view C, G equatorial view D, H exine ornamentation of equatorial view.
Figure 3 . 3 Figure 3. Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng A habitat B, C habit D, E raceme F (leaf adaxial) G (leaf abaxial) H flowers I bracteoles J calyx K standard and part of diadelphous stamen L wing M keel N gynoecium O diadelphous stamen P pod Q joint of pod R seed. Voucher A, D-H Chih Y.Chang 3620 (TCF) B, L-R C.M.Wang 17247 (TNM).
Figure 4 . 4 Figure 4. Line drawings of Smithia C.M.Wang, Chih Y.Chang & Y.H.Tseng A habit B leaf (abaxial) C stipule D flower E bracteoles F calyx G standard H wing I keel J gynoecium K diadelphous stamen L pod M joint of pod N seeds.
Figure 5 . 5 Figure 5. Distribution map of Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng.
Figure 6 . 6 Figure 6. Holotype of Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng.
Specimens examined. Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng TAI-WAN. New Taipei City: Sanxia District, "Ryoenpo" [Lungpuli], 24 Nov. 1910, 36 m alt., T.Kawakami s. n. (TAI!); Taoyuan City: "Toen" [Toyen], 25 May 1930, 134 m alt., S.Suzuki 2627 (PH, TAI!); same loc., 17 Dec. 1933, 125 m alt., S.Suzuki 4041 (TAI!); Daxi District, "Taikei" [Tahsi], 31 Mar. 1940, 119 m alt., T.Nakamura 4311 (TAI!); Luzhu District, "Toen-Nankan" [Taoyuan-Nankan], 23 Nov. 1931, 147 m alt., T.Suzuki 7876 (TAI!); Hsinchu County: Chupei City, Lienhua Temple, 19 June 1986, 63 m alt., T.C.Huang 12692 (TAI!); same loc., 24 Oct. 1996, 57 m alt., C.C.Huang 1619 (TAIE!); same loc., 30 Aug. 1996, 56 m alt., K.C.Yang 4969 (HAST!); same loc., 16 Oct. 1997, 56 m alt., Y.C.Kao 93 (HAST!); same loc., 29 Nov. 1997, 56 m alt., W.C.Leong 667 (HAST!); same loc., 15 Sept. 1998, 68 m alt., S.C.Liu 84 (TAIF!); same loc., 23 Aug. 1999, 56 m alt., C.IPeng 17683 (HAST!); same loc., 30 Aug. 1996, 57 m alt., K.C.Yang 4969 (TNM!); same loc., 15 Sept. 1998, 57 m alt., S.C.Liu 84 (TNM!); Miaoli County: Houlong Township, "Koryu" [Houlung], 1 Nov. 1924, 6 m alt., Y.Simada 1337 (HAST!, TAI!); Tunghsiao Township, Tunghsiao Township 14 th Cemetery, 27 Oct. 2016, 93 m alt., T.C.Hsu 8660 (TAIF!); same loc., 21 Oct. 2017, 103 m alt., L.H.Yang 908 (TAIE!); same loc., 2 Dec. 2017, 103 m alt., R.P.Hsieh 49 (TAIE!); same loc., 26 Sept. 2019, 93 m alt., T.C.Hsu 12070 (TAIF!); same loc., 20 Oct. 2021, 103 m alt., Z.X.Chang 2666 (TAIF!); same loc., 11 Dec. 2017, 81 m alt., C.M.Wang 17247 (TNM); same loc., 21 Oct. 2017, 81 m alt., L.H.Yang 910 (TAIE!); same loc., 1 Mar. 2018, 81 m alt., M.Y.Shen 5542 (TAIE!); same loc., 13 Oct. 2021, 81 m alt., M.Y.Shen 6841 (TAIE!); same loc., 17 Dec 2021, Chih Y.Chang 3620, 3621, 3622 (TCF); Zhunan Township, "Kityo" [Chiting], 3 Aug. 1940, 16 m alt., Fukuya s. n. (TAI!); Taichung City: "Taityushi" [Taichung], Oct. 1905, 75 m alt., G.Nakahara s. n. (PH); same loc., 27 Aug. 1931, 75 m alt., S.Suzuki 8217 (TAI!); Dajia District, Mt. Tiehchen, 12 Oct. 1997, 213 m alt., S.Y.Lu s. n. (TAIF!); Kaohsiung City: Cishan District, "Banshoryo" [Chishan], 1 Nov. 1934, 36 m alt., S.Suzuki 5825 (TAI!); Pingtung County: Gaoshu Township, "Takagi" [Kaoshu], 8 Nov. 1931, 86 m alt., T.Hosokawa 3377 (TAI!); same loc., 8 Nov. 1931, 86 m alt., T.Hosokawa s. n. (TAI!); Hengchun Township, "Koshun" [Hengchun], Aug. 1915, 15 m alt., E.Matuda 1083 (TAI!); Pingtung City, "Rokkwaiseki" [Liukueitsu], 31 Oct. 1934, 21 m alt., S.Suzuki 5713 (TAI!); Hualien County: Yuli Township, "Tamazatosyo Nodyo" [Yuli], 28 Aug. 1933, 147 m alt., Y.Yamamoto 3099 (TAI!); Hualien County, Yuli Township, "Tamazato" [Yuli], 29 Aug. 1933, 123 m alt., Y.Yamamoto 3082 (TAI!); same loc., 29 Aug. 1933, 133 m alt., Y.Yamamoto 3087 (TAI!); Yuli, 11 Feb. 1975, 116 m alt., S.Y.Lu 3440 (TAIF!).
Table 1 . 1 Specimens referenced for Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng and S. sensitiva Aiton pollen morphology. Taxon Location Coordinate Altitude Date Voucher S. yehii Taiwan. Miaoli County Tunghsiao Township, 24.44718N, 81 m 17 Dec Chih Y.Chang 3620 Tunghsiao Township 14 th Cemetery 120.69563E 2021 (TCF, TNM) S. sensitiva Thailand. Chiang Mai Province Samoeng 18.87321N, 1100 m 24 Nov C.M.Wang 17941 district, Samoeng Forest 98.78213E 2018 (TNM) China. Guangdong Province Huidong 23.19310N, 220 m 8 Sept County, Gutianshan Nature Reserve 114.78134E 1984
Table 2 . 2 Summary of diagnostic characters of Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng and its similar species. Characters S. yehii S.
sensitiva (Efloraofindia 2007; Sa and Delgado-Salinas 2010; Balan and Predeep 2017) S. ciliata (Huang and Huang 1993; Sa and Delgado-Salinas 2010) Leaflet pairs (2)4-9 4-11 4-7 Leaflet size 3.5-7.0 × 1.2-2.3 mm 4-15 × 2-3 mm 6-12 × 2-4 mm Leaflets, adaxial dark green at apex, light same color between same color between color green at base when apex and base apex and base young and mature Flowers, number 1-7 3-6 12 to many per raceme Flowers 0.7-1.0 cm long 0.8-1.5 cm long c. 1 cm long Calyx entire at margin, entire at margin, scarious, with ciliate at margin, membranous, with scarious, with parallel parallel veins reticulate veins veins Corolla pale yellow vivid yellow white or yellow Style 3.4-4.1 mm long c. 8 mm long c. 2.5 mm long Pod shape and more or less straight, more or less straight, c. 0.4 cm slightly orbicular, 1-1.5 cm long size 0.5-0.8 cm long long Jointed number (4)6-7 4-6 6-8 of pod Distribution endemic to Taiwan, widely distributed in Australia, widely distributed in Taiwan, China, in wetlands and open India, Madagascar and Tropical Bhutan, India, Japan, Malaysia, Nepal, places, at elevations of Asia, in field margins, wetlands; Philippines, Thailand and Vietnam. < 300 m at elevations of < 1000 m Taiwan, in margin of thickets, at elevation of 1,000-1,800 m (3.4-4.1 mm) than S. sensitiva (c. 8 mm) (Efloraofindia 2007; Sa and Delgado-Salinas 2010) (Table
Table 3 . 3 Comparison Taxon Polar axis Equatorial axis P/E ratio Exine Murus wide Perforate size length (μm) length (μm) ornamentation (μm) (μm) S. yehii 21.2±1.0 19.5±1.6 1.1±0.1 perforate 0.5±0.1 0.4±0.1 (19.6-22.7) (16.5-21.9) (0.9-1.3) (0.3-1.1) (0.2-0.6) S. sensitiva 21.0±1.4 18.4±1.6 1.1±0.1 perforate 0.4±0.1 0.2±0.1 (18.5-23.5) (15.9-20.4) (0.9-1.3) (0.3-0.7) (0.1-0.3) of pollen characters of Smithia yehii C.M.Wang, Chih Y.Chang & Y.H.Tseng and S. sensitiva Aiton. Key to Smithia yehii and its similar species (modified from Huang and Huang (1993) , and Sa and Delgado-Salinas (2010) 1 Inflorescences often with more than 12 flowers, calyx ciliate at margin, membranous, with reticulate veins; pods slightly orbicular, more than 1.1 cm long ............................................................................................... S. ciliata -Inflorescences with fewer than 7 flowers, calyx entire at margin, scarious, with parallel veins; pods more or less straight, less than 1 cm ..............................2
Table 4 . 4 Students' t scores and p values for quantitative characters of pollen grains. Characters t score p value Polar axis long -1.753 0.095 Equatorial axis long -1.687 0.107 P/E ratio 0.227 0.823 Interval between perforations -2.076 0.044* Perforation size -7.361 0.000* Note: p value significance: *p < 0.05, p < 0.01, *p < 0.001
|
10.1073/pnas.1503926112
|
public-domain
| null | null |
openalex
|
Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits. O ver half a century ago, Ehrlich and Raven (1) coined the term 'coevolution' and proposed that coevolutionary interactions between species with close ecological relationships generated much of the eukaryotic biodiversity on Earth. One of their primary examples of coevolution was the chemically mediated interactions between butterflies of the subfamily Pierinae (Pieridae, Lepidoptera) and their angiosperm host-plants in the order Brassicales. Members of the plant order Brassicales are united by their production of secondary metabolites called glucosinolates (i.e., mustard oils). Upon tissue damage, glucosinolates are modified into toxins long studied for their defensive properties and flavor (e.g., mustard and horseradish) (2) . In the Arabidopsis thaliana (thale cress) genome, at least 52 genes are involved in glucosinolate biosynthesis (3, 4) and some exhibit strong evidence of adaptive evolution that is attributed to herbivore mediated selection (5, 6) . Pierinae caterpillars detoxify the glucosinolates of their Brassicales host-plants by redirecting these otherwise toxic breakdown products to inert metabolites using a gene that encodes a nitrile-specifier protein (7) . The key innovation of the Brassicales, defensive glucosinolates, evolved roughly 90 million years ago (Ma); within 10 million years, Pierinae responded with their own key innovation, the nitrilespecifier protein, and colonized the Brassicales. Subsequently, Pierinae net diversification rates increased compared with that of their sister clade Coliadinae, whose members did not colonize Brassicales (8) . Although these studies provide "perhaps the most convincing example" that the evolution of a key innovation resulted in an increased net diversification rate (9), much remains unknown about the origins and subsequent evolutionary dynamics of the key innovations that have had macroevolutionary consequences. To address this gap in the literature, here we further investigate these key innovations in the aforementioned plant and butterfly lineages by (i) assessing if these innovations increased in complexity over time and are associated with shifts in net diversification rates in both coevolutionary partners, (ii) identifying genomic mechanisms that facilitated the appearance and escalation of innovations that mediated the observed coevolutionary dynamics, and (iii) testing
Significance This research uncovers the mechanisms of an ancient arms race between butterflies and plants, seen today in countless gardens as caterpillars of cabbage butterflies that devour cabbage crop varieties. Nearly 90 million years ago, the ancestors of Brassica (mustards, cabbage) and related plants developed a chemical defense called glucosinolates. While very toxic to most insects, humans experience glucosinolates as the sharp taste in wasabi, horseradish and mustard. Here we report that this triggered a chemical arms race that escalated in complexity over time. By investigating the evolutionary histories of these plants and insects, we found that major increases in chemical defense complexity were followed by butterflies evolving countertactics to allow them to continue to attack and feed on the plants. A common temporal scale is provided between the two chronograms. The branches in the Brassicales phylogeny are colored to indicate the origin of indolic glucosinolates (purple), methionine derived glucosinolates (green), and novel structural elaborations to glucosinolates unique to the core Brassicaceae lineage (orange). Vertical dashed lines also indicate the origin of these novel chemical groups. Primary host-plant associations of various Pierinae lineages are similarly colored: orange (Brassicaceae), green (Capparaceae or Cleomaceae), orange-green (mixture of previous), purple (more basal Brassicales that synthesize indolic glucosinolates), blue (non-Brassicales feeding) and gray (unknown). The phylogenetic positions for the At-α and At-β WGDs are depicted with white diamond symbols, and significant net diversification rate shifts with red star symbols. whether any observed increases in net diversification rates are associated with adaptive evolution. Brassicales species synthesize more than 120 different glucosinolate compounds (10, 11) . To investigate their evolution and potential escalation, we placed this diversity into a robust phylogenetic framework (Fig. 1 ). Inferring these relationships requires significantly more data than generated in previous studies, where phylogenetic estimates lack sufficient statistical support to adequately describe the last 90 million years of Brassicales evolution (SI Appendix, Text S1) (12) . To resolve the phylogenetic backbone of Brassicales, we generated transcriptomes and identified orthologs from taxa across 14 families (average de novo assembled transcripts per species, 42,657; average length, 794 bp), and combined these with orthologs from nine available genomes (SI Appendix, Text S1). A time-calibrated phylogenomic tree was then generated and family level glucosinolate diversity and species richness were mapped onto it to identify the evolutionary points of origin for novel glucosinolate groups and shifts in net diversification rates (i.e., speciation minus extinction rates) (Fig. 1 ). We also localized the two ancient whole genome duplications (i.e., WGD or polyploid events) of Brassicales, named At-β and At-α (Fig. 1 ) (13), using further analyses of age distribution of gene duplicates, assessment of their gene trees, and a comparison of genomes (SI Appendix, Text S2). The exact phylogenetic placement of both the At-β and At-α has been a mystery due to a lack of a robust phylogenetic framework and a dearth of genomic resources from the vast majority of families in Brassicales. In addition, we performed evolutionary analyses of the core biosynthetic pathways across Brassicales families to investigate the origin of pathways that encode novel glucosinolate chemical classes (Fig. 2 and SI Appendix, Text S3). Similarly, we assembled a dataset from eight gene regions (6,716 bp) and 96 specimens to estimate the divergence times and diversification dynamics of the major lineages of Pieridae butterflies on a phylogeny, which has recently been well resolved at the genus level ( 14 ) (SI Appendix, Text S4). To investigate the evolutionary dynamics of the nitrile-specifier protein gene (i.e., counter adaptation against glucosinolates), we surveyed nine phylogenetically important species using transcriptome and whole genome sequencing (Fig. 2 and SI Appendix, Text S5) and tested the activity of identified nitrile-specifier proteins for two pivotal genera (Anthocharis and Pieris) using glucosinolate-myrosinase assays on gut extract and expressed proteins. Our results identify three important steps of escalation in the evolution of Brassicales that are reflected in ancestral Pierinae feeding patterns (Fig. 1 ). Brassicales, which appeared 92 Ma (median crown age; 95% HPD 50-133 Ma), initially were only able to synthesize glucosinolates from phenylalanine and branched chain amino acids (Fig. 2B ; ancestral pathway). After the At-β WGD event 77.5 Ma (95% HPD 42-112 Ma), indolic glucosinolates appeared, which are synthesized from the amino acid tryptophan. Use of this novel substrate, as well as the previous ones, is restricted to the descendant families of the At-β event. These families show a near uniform retention of gene duplicates across the beginning of the core pathway, whose biosynthetic steps involve the conversion of either tryptophan or one of the more ancestral amino acid substrates into an intermediate aldoxime compound. No such retention of duplicates is seen in other metabolic pathways (SI Appendix, Text S3). Rather than simply functioning as redundant copies, the retained gene duplicates of the glucosinolate core pathway, including the cytochrome P450 79 (CYP79) step, have distinct substrates and products that are independently regulated by their own transcription factors (i.e., they have nonoverlapping functions) (SI Appendix, Text S3) (15) . Here, we infer that the ability to synthesize glucosinolates from these aldoxime products is the ancestral state for Brassicales. This conclusion is supported by a recent analysis of the CYP79 family in a distant relative (Poplar; Populus trichocarpa), which revealed that this gene family in other plant orders is involved in the biosynthesis of herbivore-induced volatile aldoximes from phenylalanine and branched chain amino acids (16) . Our results further indicate that the escalation of glucosinolate diversity, using new amino acid substrates to produce novel classes of compounds used as defenses in the arms-race against the butterflies, occurred via both single gene and whole genome duplications, involving the retention and neofunctionalization of both regulatory and core biosynthesis genes. Nearly all Brassicales-feeding Pierinae butterflies consume families of Brassicales that synthesize indolic glucosinolates, and these compounds are well documented as important cues for host recognition and oviposition. Our mean estimate of the colonization of Brassicales by Pierinae butterflies is 68 Ma (Pierinae crown age; 95% HPD 60-75 Ma), concordant with the appearance of glucosinolate detoxification and an increase in their diversification rate (8) . Together these insights provide a more detailed understanding of the colonization dynamics of Brassicales than previously described ( 8 ) by associating the diversification of Pierinae not with the timing of the origin of Brassicales per se, but rather with the origin of indolic glucosinolates. Indolic glucosinolates elicited an evolutionary response in Pierinae that highlights a dynamic identified by Erhlich and Raven (1) , where a formerly toxic compound becomes an attractant after colonization. A second major phase of escalation occurred when the ancestors of the plant lineages Capparaceae and Cleomaceae evolved a new suite of glucosinolate compounds derived from another novel substrate (methionine) (Fig. 1 ). This escalation in glucosinolate diversity occurred again via gene duplications followed by the neofunctionalization of both regulatory and core biosynthesis genes (Fig. 2 and SI Appendix, Text S1) and coincides with a significant increase in net diversification rate near the origin of these plant families (P value < 0.01) (Fig. 1 and SI Appendix, Text S1). Although the Pierinae that colonized the Brassicales necessarily fed on plants containing indolic glycosinolates (Fig. 1 ), today there are significantly fewer species feeding on the indolic-only lineages compared to the Capparaceae and Cleomaceae (30 vs. 133 species, respectively, P value < 0.0001). The final escalation event we discovered appeared 32 Ma (95% HPD 17-46 Ma) with the evolution of Brassicaceae (mustard family), which have the greatest diversity of glucosinolates within Brassicales. We find this glucosinolate diversity to be derived from genes originating via tandem duplications and retention following the second major WGD of Brassicales, the At-α event (17, 18) . This chemical defense escalation coincides with a dramatic increase in Brassicaceae diversification (P value < 0.01; Fig. 2 ; SI Appendix, Text S1). Shortly after Brassicaceae appeared, two independent lineages of Pierinae herbivores colonized them: one from the Anthocharidini and one from the Pierina butterfly lineages (Fig. 1 and SI Appendix, Text S4). Both butterfly colonizations coincide with increased diversification rates (P value < 0.01) (SI Appendix, Text S5). Thus, we have uncovered a dynamic escalation process that appears to have affected the macroevolution of Brassicales plants and their Pierinae herbivores. This process was facilitated by gene and genome duplications in Brassicales. Finally, comparison of macroevolutionary patterns of both groups shows a striking temporal concordance consistent with the expected outcome of diffuse coevolutionary interactions (Fig. 2 ). Having established that Brassicaceae harbor the greatest glucosinolate diversity among plants and that this diversity arose via gene and genome duplications, we investigated whether butterfly detoxification mechanisms reflected a corresponding escalation in trait complexity. All of the Brassicales-feeding Pierinae could be using the same detoxification gene, evolving via allelic turnover, or there could be different gene copies having independent functional roles (e.g., specific to different types of glucosinolates). The latter condition would be evidence of an escalation at the molecular level via gene-family dynamics. Our results show that rather than every butterfly using the same nitrile-specifier gene for glucosinolate detoxification, species have lineage-specific copies derived from a combination of at least four paralogous clades that differ in the glucosinolates they detoxify (Fig. 2 ). Of the two independent colonizations of Brassicaceae by Pierinae, the Anthocharidini lineage exemplar species (Anthocharis cardamines) has two nitrile-specifier genes derived from a single clade; they differ by 16 amino acids and their nitrile-forming activity against different glucosinolates is different (Benzyl: ancestral compound and 4-methylsulfuinylbutyl: novel methionine derived compound; SI Appendix, Text S5). In the Pierina exemplar species (Pieris rapae), there are two nitrile-specifier genes, but they come from different gene family clades. Although nitrile-forming activity is found in the paralogous clade not found in A. cardamines, similar nitrile-forming activity could not be detected in the gene from the paralogous clade most closely related to the A. cardamines clade (SI Appendix, Text S5). Thus, not only do different nitrile-specifier gene lineages exhibit functional differences against glucosinolates, but the two independent colonizations of the Brassicales were facilitated by independent nitrile-specifier gene lineages. We further assessed the relationship between nitrile-specifier genes and hostplant glucosinolate content by investigating how these detoxification genes evolved after butterfly lineages colonized plants without a glucosinolate defense system (Fig. 1 ). Aporini stopped using Brassicales 37 Ma (95% HPD 34-42 Ma) and in that lineage's exemplar, Delias nigrina, neither nitrile-specifier genes were found in its genome nor any detectable enzymatic activity in its larval midgut (SI Appendix, Text S5). Thus, the two independent lineages that colonized Brassicaceae used independent copies of the nitrile-specifier protein, and feeding upon glucosinolate-containing plants appears necessary to maintain members of the nitrile-specifier gene family within a butterfly genome. Our genomic data also provides a means of testing whether these patterns of escalation are associated with neutral or adaptive evolution. In Pierinae, we asked whether evolutionary dynamics of the nitrile-specifier protein departed from neutrality in the lineages leading to the two independent functional loci in Anthocharidini and Pierina. Using a maximum likelihood analysis of codon evolution (site-branch analyses), we found evidence of greater than expected levels of amino acid substitutions (i.e., diversifying selection) in both lineages (P value < 0.0001; SI Appendix, Text S5), consistent with adaptive evolution. In Brassicales, we asked whether species diversification in Brassicaceae was driven due to a neutral, random process (i.e., reciprocal gene loss) (19) or an adaptive process (i.e., facilitated by the origin of a novel trait) following the At-α event (Fig. 1 and SI Appendix, Text S6). Reciprocal gene loss is a mechanism of speciation, which has led to the emergence of new yeast species following whole genome duplication (19) , that involves the passive loss of alternate copies of duplicated genes among different populations leading to their reproductive isolation (20) . Because the At-α event predates the split between the Arabidopsis (4) and Aethionema (21) lineages, by quantifying the genome-wide duplication status of these earliest diverging Brassicaceae we can test whether reciprocal gene loss is associated with the increased diversification rates seen in the Arabidopsis lineage, which has 3615 species compared with 45 species in the Aethionema lineage. Of At-α duplicate genes, both lineages share the duplication status for 74% (53% reverted to singletons, 21% retained as duplicates), whereas the Aethionema and Arabidopsis lineages had 18% and 8% of these genes uniquely lost, respectively. Thus, lineage specific loss of duplicates was inversely correlated with shifts in diversification rates, and a putative genome-wide level of reciprocal gene loss was estimated at less than 0.4%. We conclude that reciprocal gene loss following the At-α event was therefore not a major contributor to diversification, as the vast majority of genes quickly reverted to single copy status after the At-α event and only a subset of genes, such as those in the glucosinolate pathway were retained as duplicates. Consistent with the hypothesis that retention of duplicates after WGD is driven by selective benefits (22) , previous analyses indicates a high metabolic cost of glucosinolate production (23) , a result incompatible with the retention of glucosinolate duplicate being neutral. Half a century ago, Ehrlich and Raven (1) hypothesized that plant-insect interactions driven by diffuse coevolution over long periods of evolutionary time could be a major source of terrestrial biodiversity. Here, our analyses find strong support for this hypothesis the repeated escalation of key innovations and bursts of diversification in Brassicales plants and Pierinae butterflies over the past 80 Myr. Importantly, gene and genome duplication events, via birth-death dynamics, have had a central role in these macroevolutionary events on each side of the plant-insect interaction. Although gene birth-death dynamics are known to be important in immune response and xenobiotic interactions (24, 25) , our observation of similar dynamics in the nitrile-specifier gene family of butterflies provides a much needed data point from a novel gene family. These observed gene birth-death dynamics suggest that allelic change within genes may not be sufficient for long-term success during direct, as well as diffuse, coevolutionary interactions, extending Ohno's seminal idea on the role of gene duplications in macroevolution (26) , where evolutionary novelty more often occurs by gene duplication rather than by allelic variation of existing genes. The birth-death dynamics of certain gene families likely also drives the coevolutionary interactions described in other well studied host plant and herbivore systems (27) , including between Apiaceae (Parsley family) and Papilionidae butterflies (28) or Oecophoridae moths (29) . Interestingly, the cytochome P450 gene family appears to also play a crucial role in these systems as in the Brassicales. For example, the biosynthesis of furanocoumarins in Apiaceae but also the detoxification of these compounds by both Papilionidae and Oecophoridae involve cytochrome P450s (27) . Rather than identifying patterns that are unique to these clades, we take these findings to suggest that such gene and genome duplication dynamics may be a general feature of the coevolutionary interactions that generated much of the diversity on Earth.
Materials and Methods Transcriptomes were sequenced, assembled de novo across Brassicales, and combined with available plant genomes to: (i) Resolve phylogenetic relationships among families using 1155 single copy genes and divergence dates estimated with fossil calibrations (SI Appendix, Text S1); (ii) detect and phylogenetically localize two ancient whole genome duplications (SI Appendix, Text S2); and (iii) investigate the origin and evolution of glucosinolate pathways (SI Appendix, Text S3). We identified shifts in speciation rates following the origin of these traits (SI Appendix, Text S1), where chemical complexity appears to have escalated over time, and that Brassicalesfeeding Pieridae butterflies have evolved counteradaptations from duplicate genes and coradiated with plant hosts. Parallel phylogenetic and temporal estimates of Pieridae used 7 nuclear genes and 1 mitochondrial gene, with divergence dates estimated with fossil calibrations (SI Appendix, Text S4). Pierinae species diversity and host-plant use were based on existing databases (SI Appendix, Text S4). The evolutionary dynamics of the nitrilespecifier protein were reconstructed using whole genome and transcriptome sequencing coupled with sequence analysis of codon evolutionary dynamics, with functional performance assayed on heterologously expressed proteins using established methods (SI Appendix, Text S5). Lastly, comparative genomic analyses were conducted to investigate whether observed net diversification rate shifts could be associated with neutral gene loss events following the most recent whole genome duplication at the base of Brassicaceae (SI Appendix, Text S6). coevolution | phylogenomics | evolutionary novelty | chemical defenses | diversification
Fig. 1 . 1 Fig. 1. Phylogeny and diversity. A chronogram of both Brassicales families (Upper) and Pierinae butterfly genera (Lower), showing species numbers and identification of clades in the adjacent table.A common temporal scale is provided between the two chronograms. The branches in the Brassicales phylogeny are colored to indicate the origin of indolic glucosinolates (purple), methionine derived glucosinolates (green), and novel structural elaborations to glucosinolates unique to the core Brassicaceae lineage (orange). Vertical dashed lines also indicate the origin of these novel chemical groups. Primary host-plant associations of various Pierinae lineages are similarly colored: orange (Brassicaceae), green (Capparaceae or Cleomaceae), orange-green (mixture of previous), purple (more basal Brassicales that synthesize indolic glucosinolates), blue (non-Brassicales feeding) and gray (unknown). The phylogenetic positions for the At-α and At-β WGDs are depicted with white diamond symbols, and significant net diversification rate shifts with red star symbols.
Fig. 2 . 2 Fig. 2. Evolution of glucosinolate biosynthethic pathways and detoxification nitrile-specifier protein (NSP) family. (A) Shifts in diversification rates during Brassicales evolution are highly punctuated (Upper). Lines are colored identical to lineages in Fig. 1. (Lower) How Pierinae butterflies have diversified during the same period. Time estimates are shown at the bottom. (B) An illustration of the evolution of core glucosinolate pathways across Brassicales; with substrates tryptophan (Trp), phenylalanine (Phe), and methionine (Met) shown at the top, enzymes depicted as white ovals, and each pathway as black vertical lines. (C) Photographs show Pieris brassicae (Upper) and Pieris rapae (Lower) caterpillar feeding on Brassica oleracea (European cabbage). (D) Evolution of the NSP gene family is shown across select Pierinae genera, indicating the birth and death dynamics of four paralogous clades.
| www.pnas.org/cgi/doi/10.1073/pnas.1503926112 Edger et al.
| www.pnas.org/cgi/doi/10.1073/pnas.1503926112 Edger et al.
|
10.1186/s12906-020-2818-8
|
cc-by
| null | null |
openalex
|
Background: Candida vaginitis is a global health hazard that increases morbidity among women of childbearing age. Recent studies have revealed a high incidence of drug-resistant Candida strains. Additionally, treating Candida vulvovaginitis during pregnancy is challenging as antifungal therapy is associated with fetal abnormalities. Hence, it is important to develop novel therapeutic strategies to treat vulvovaginal candidiasis. Methods: In this study, we used the disc diffusion method to evaluate the anticandidal activity of different Syzygium aromaticum extracts (methanol, ethyl acetate, n-hexane, and diethyl ether) against C. albicans, C. glabrata, and C. tropicalis. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis of different S. aromaticum extracts was performed to determine active components exhibiting anticandidal activity. Cytotoxicity of different clove extracts against the HUH7 cell line was evaluated. Results: The ethyl acetate extract exhibited the highest antifungal activity against C. albicans, C. glabrata, and C. tropicalis with inhibition zone diameters of 20.9, 14.9, and 30.7 mm, respectively. The minimum inhibitory concentration of the S. aromaticum ethyl acetate extract was 250 μg/disc against C. tropicalis, and 500 μg/disc against C. albicans and C. glabrata, while the minimum fungicidal concentration was 0.5 mg/disc against C. tropicalis and 1 mg/disc against the C. albicans and C. glabrata. GC-MS analysis of the ethyl acetate extract revealed the main bioactive compound as eugenol (58.88%), followed by eugenyl acetate (23.86%), transcaryophyllene (14.44%), and α-humulene (1.88%). The cytotoxicity assay indicated that the diethyl ether extract demonstrated the lowest toxicological effect against the HUH7 cell line, with a relative IC 50 of 62.43 μg/ml; the methanolic extract demonstrated a higher toxicity (IC 50 , 24.17 μg/ml). Conclusion: As the S. aromaticum extract exhibited high antifungal activity at low concentrations, it can be a potential source of natural antifungal drugs.Background Candidal vulvovaginitis is a common inflammatory disease among women, caused by an infection of the Candida species, especially C. albicans . Candida vaginitis is characterized by vaginal discharge, pruritus, itching, dyspareunia, and erythematous vulva . Epidemiological studies have indicated that C. albicans (70.0-89.0%) is the main causative agent of Candida vaginitis, followed by C. glabrata (3.4-20.0%) . The high incidence of infection rates among pregnant women may be attributed to the high secretion of sex hormones during pregnancy [7, 8] . A prescription of antifungal drugs during pregnancy is a challenging task as antifungals are accompanied by possible fetal toxicity and teratogenicity . The virulence factors of the Candida species including hyphae formation, production of extracellular enzymes, adhesion, and biofilm formation, help in fungal colonization in the host and the establishment of fungal infection in the vagina . Candida pathogens adhere to the epithelial cells of the vagina to initiate fungal infection through the production of proteins called adhesins . Moreover, the formation of C. albicans pseudomycelium enhances the ability of the fungus to invade the host vaginal tissues . Additionally, Candida species secrete several extracellular enzymes such as lipases, phospholipases, and hemolysins that aid in the adhesion and host tissue invasion . In the USA, C. glabrata is the second most common causative agent of invasive candidiasis (12-18%), reportedly exhibiting resistance against fluconazole . An earlier study reported that approximately 94% of all C. albicans isolates from vaginitis patients exhibit resistance to fluconazole . Furthermore, another study has reported that certain Candida isolates exhibit resistance to fluconazole and econazole . Syzygium aromaticum (clove) is an aromatic medicinal plant that belongs to the Myrtaceae family . Reportedly, clove oil possesses antifungal, antibacterial, antiviral, and insecticidal properties due to the presence of phytoactive compounds such as eugenol, eugenyl acetate, and β-caryophyllene . The antifungal activity of S. aromaticum has been demonstrated by Khan et al. (2009) , who reported that the ethanolic extract of clove was highly effective against C. albicans with a Minimum inhibitory concentration (MIC) value at 156 μg/mL . The same result has been confirmed by Gonelimali et al. (2018) and Sahal et al. (2019) , who detected the potency of clove extracts against C. albicans and C. tropicalis strains at concentrations of 20% w/v and 500 μl/mL, with inhibition zones diameter of 25.2 and 28 mm, respectively [23, 24] . The high incidence of vaginal candidiasis among pregnant women, in addition to the emergence of resistant Candida strains to different antifungal agents, enhances the necessity to formulate novel and safe natural therapeutic agents. Hence, this study aimed to investigate the anticandidal activity of different S. aromaticum extracts against three Candida species.
Methods
Preparation of plant extracts The extraction procedure was performed using different organic solvents (methanol, ethyl acetate, diethyl ether, and n-hexane), demonstrating different polarities to allow the extraction of all hydrophilic and lipophilic bioactive compounds. The flower buds of S. aromaticum were obtained from a local market in Riyadh, Saudi Arabia. We identified the plant material and the identification was confirmed by the Saudi herbarium, Botany Department, College of Science, King Saud University. The plant material was deposited at the herbarium with a voucher number (KSU-14682). Clove buds were disinfected using 0.5% sodium hypochlorite solution (NaOCl), washed three successive times using sterile distilled water, and allowed to dry. The dried plant material was homogeneously powdered using a mechanical mortar. The powdered sample (15 g) was added to 200 mL of the different organic solvents and incubated on a magnetic stirrer for 48 h. Next, the mixture was centrifuged at 9000 rpm for 10 min to remove residues. The supernatant was filtered using a Whatman filter paper to obtain a clear filtrate. The filtrate was concentrated using a rotatory evaporator and stored at 4 C until use. The yield of the extract was calculated using the following formula: Percentage extract yield 1⁄4 R=S ð ÞÂ100 where R is the weight of the residue of the plant extract; S is the raw plant sample weight.
Candida isolates Three Candida species, C. albicans, C. tropicalis, and C. glabrata were obtained from King Khalid Hospital, Riyadh, Saudi Arabia. The fresh inoculum was prepared by subculturing each Candida species onto Sabouraud dextrose agar (SDA) medium at 35 C for 48 h.
Preparation of candidal inoculum SDA slants were prepared and inoculated with different Candida species. The fungus was harvested using 5 mL sterile saline solution. The absorbance of the fungal suspension was measured at 560 nm using a spectrophotometer and the cell count was adjusted to attain a viable cell count of 10 7 CFU/mL for each Candida species.
Anticandidal bioassay The anticandidal activity of different S. aromaticum extracts was evaluated by the disc diffusion method. The anticandidal bioassay was performed to evaluate the antifungal potency of the extract, in which 10 mL SDA medium was poured into the sterile petri dishes as a basal layer, followed by the addition of 15 mL seeded medium previously inoculated with the prepared microbial suspension (1 mL of fungal suspension/100 mL of medium) to attain a viable cell count of 10 5 CFU/mL. The sterilized filter paper discs (diameter 8 mm) were loaded with 10 mg of different clove extracts and placed over the seeded plates after solidification [25, 26] . Terbinafine was used as a positive control at a concentration of 50 μg/disk according to CLSI guidelines. Interpretation criteria of terbinafine as the antifungal agent corresponding to the inhibition zone diameter was as follows: >= 20 mm, sensitive; 12-19 mm, dose-dependent; <= 11 mm, resistance . Eugenol (Sigma-Aldrich, USA), the main active constituent of clove, was also used as a standard phytoactive compound. Sterile filter paper discs loaded with 50 μg of eugenol were placed over the seeded plates . The plates were incubated at 4 C for 2 h to allow the diffusion of the clove extracts throughout the medium. The plates were further incubated at 35 C for 48 h and the inhibition zone diameter was measured using a Vernier caliper as an indication of antifungal activity.
Determination of minimum inhibitory concentration (MIC) MIC was defined as the lowest concentration of plant extract exhibiting antifungal activity. MIC was evaluated only for the ethyl acetate extract of S. aromaticum as the most effective clove extract. SDA medium (15 mL) was poured into a sterile petri dish as a basal layer, followed by the addition of 10 mL seeded medium (as described above), and allowed to solidify. Sterile filter paper discs (diameter 8 mm) loaded with different concentrations of S. aromaticum ethyl acetate extract (0.125, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/disc) were placed over the seeded medium. The plates were incubated at 5 C to allow extract diffusion. The plates were then incubated at 35 C for 48 h and the inhibition zone diameter was measured using a Vernier caliper. The lowest concentration that exhibited antifungal activity against Candida species was recorded as the MIC.
Determination of minimum fungicidal concentration Minimum fungicidal concentration (MFC) was defined as the lowest concentration of the clove extract exhibiting fungicidal activity. Streaks obtained from the inhibition zone area of MIC and two successive concentrations were cultured on freshly prepared SDA plates. The plates were incubated at 35 C for 48 h and the lowest concentration that exhibited no fungal growth was recorded as MFC.
Gas chromatography-mass spectrometry (GC-MS) analysis of S. aromaticum extracts Phytochemical analysis of the S. aromaticum extracts was performed by GC-MS for the detection of active compounds exhibiting antifungal activity. The GC-MS analysis was performed using the GC-MS Thermo Trace GC Ultra / TSQ Quantum GC. The phytochemical analysis was performed using a TR5-MS capillary column, (30 m × 0.25 mm; 0.25 μm film thickness). The oven was programmed to a ramp rate of 6 C/min to increase the temperature from 40 to 200 C. The operating conditions were as follows: helium as a carrier gas with a flow rate of 1 mL/min, injector and detector temperatures were 250 C, split ratio was 1:50. The conditions for mass spectrometry were as follows: mass range from m/z, 40-400 amu; ionization potential 70 eV; electron multiplier energy 2000 V. The chemical constituents of the clove bud extracts were identified by comparing the results of the GC-MS analysis with the reference retention time and spectral mass data of the NIST database.
Cytotoxicity assay The human hepatoma (HUH7) cell line was obtained from the Zoology department, College of Science, King Saud University, Saudi Arabia. The toxicity of different clove extracts of diethyl ether, ethyl acetate, methanol, and n-hexane against the HUH7 cell line was evaluated using the 3-(4,5-dimethylthiazol)-2,5diphenyl tetrazolium bromide (MTT) assay . The cells were cultured in minimal essential medium (Sigma-Aldrich, USA) supplemented with 0.1% gentamicin (Virbac) and 5% fetal calf serum (Adcock-Ingram), incubated in a 5% CO 2 incubator. HUH7 cells were inoculated in 96-well plates and incubated at 37 C for 24 h in an 5% CO 2 incubator for cell adherence to the plate. The crude clove extracts were redissolved in methanol (10 mg/mL), and appropriate dilutions were prepared. Cells were treated with the extracts of concentrations ranging from 0.0065 to 1 mg/ml. After treatment for 48 h, the supernatant was removed and the developing solution (MTT) was added at a concentration of 5 mg/mL to the wells for the formation of formazan crystals. The plates were incubated at 37 C for 4 h and supernatants were removed. Finally, 50 μL of dimethyl sulfoxide (DMSO) was added to the wells, stabilizing the formed formazan crystals. Absorbance of the soluble formazan in plates was measured at a wavelength of 570 nm. The absorbance corresponding to the concentration inducing a 50% inhibition of cell viability (IC 50 ) was calculated.
Statistical analysis The susceptibility of Candida to different clove extracts was analyzed with GraphPad Prism 5.0 (Graph-Pad Software, Inc., La Jolla, CA, USA) using one-way analysis of variance and Tukey's test. The data are presented as mean ± standard error for triplicates. The data were considered statistically significant when the P-value was less than 0.05.
Results
Extract yield The highest S. aromaticum yield (6.09%) was obtained using diethyl ether, followed by methanol (4.67%), n-hexane (3.02%), and ethyl acetate (2.57%).
Anticandidal activity Screening the antifungal activity of different
MIC and MFC of S. aromaticum ethyl acetate extract The MIC of S. aromaticum ethyl acetate extract against C. tropicalis was 0.25 mg/disc, with an inhibition zone tropicalis the most sensitive to this clove extract (Fig. 1 ). The MFC of the S. extract against C. tropicalis was 0.5 mg/disc as no growth of Candida was observed at this concentration. Similarly, no C. albicans and C. glabrata growth was observed at 1 mg/disc.
Gas chromatography-mass spectrometry analysis (GC-MS) of S. aromaticum extracts GC-MS analysis of S. aromaticum ethyl acetate extract revealed that eugenol (58.88%) was the most abundant active component, followed by eugenyl acetate (23.86%), trans-caryophyllene (14.44%), αhumulene (1.88%), caryophyllene oxide (0.74%), and longipinocarvone (0.19%) (Table 3 and Fig. 2 ). Furthermore, the main bioactive component of the diethyl ether extract was eugenol (66.48%), followed by eugenyl acetate (31.73%), trans-caryophyllene (1.63%), caryophyllene oxide (0.31%), and α-humulene (0.21%) (Table 4 and Fig. 3 ). In contrast, new phytochemical constituents detected in the S. aromaticum methanolic extract included musk ketone (2.78%), αcubebene (0.79%), chavicol (0.78%), β-cadinene (0.59%), and α-farnesene (0.37%). Other constituents such as eugenol (55.58%), eugenyl acetate (19.83%), trans-caryophyllene (15.71%), α-humulene (2.46%), and caryophyllene oxide (1.10%) were also detected (Table 5 and Fig. 4 ). Similarly, eugenol (61.37%) was the most abundant bioactive component in the nhexane extract (Table 6 and Fig. 5 ).
Cytotoxicity assay The diethyl ether clove extract demonstrated the lowest toxic effect against the HUH7 cell line, with a relative IC 50 of 62.43 μg/ml, while higher toxicity of was detected with the methanolic extract, with an IC 50 of 24.17 μg/ml. Moreover, the n-hexane and ethyl acetate extracts of clove exhibited moderate toxicity against HUH7 cells with relative IC 50 values of 42.19 and 33.68, respectively (Fig. 6 ).
Discussion All extracts of S. aromaticum exhibited antifungal activity against the concerned candidal strains evaluated in this study (Table 1 ). These results concurred with those of Mansourian et al., demonstrating that S. aromaticum extracts (10 mg/100 μl) possessed antifungal activity against C. albicans, with an inhibition zone diameter of 29.6 mm . Reportedly, clove extracts possess antioxidant and antimicrobial properties due to the presence of phenolic compounds, such as flavonoids, hydroxybenzoic acids, and hydroxyphenyl propenes . Phytochemical analysis of the S. aromaticum ethyl acetate extract demonstrated that eugenol was the most abundant active component (58.88%), followed by eugenyl acetate (23.86%), trans-caryophyllene (14.44%), and α-humulene (1.88%). These phytochemicals were consistent with the study by Jirovetz et al., who reported that eugenol was the most abundant active phytochemical component, constituting 76.8% of the total active compound . Additionally, similar results were reported by Chaieb et al., demonstrating that clove oil was predominantly composed of eugenol (70%), followed by eugenyl acetate (5.6%), and β-caryophyllene (1.4%) . The high efficiency of the ethyl acetate clove extract as an antifungal agent offers a potential natural anticandidal drug for the treatment of Candida vaginitis, avoiding the side effects associated with several chemotherapeutic agents used in the treatment of vaginitis. In addition, Chami et al. ascertained that clove extract constituents are a promising source for the curative and prophylactic therapy of vulvovaginal candidiasis . The inhibition zone diameters recorded with eugenol (50 μg) as a standard compound against the concerned Candida species were less than those recorded for the crude clove extract (10 mg). These results may be attributed to the high content of eugenol in different extracts, confirmed by GC-MS analysis of clove. GC-MS analysis of S. aromaticum extracts (diethyl ether, ethyl acetate, n-hexane, and methanolic) revealed that eugenol was the main active Eugenol exhibited antifungal potency against C. albicans strains, demonstrating inhibition zone diameters of 15.67 mm. This result was consistent with that of Pavesi et al., who detected the potency of eugenol at a concentration of 57 μg/disk against C. albicans, with an inhibition zone diameter of 12.1 mm . In the present study, the ethyl acetate clove extract demonstrated the highest antifungal efficacy against the different Candida species compared to the other solvent extracts evaluated. The antifungal potency of S. aromaticum could be attributed to the high eugenol content of clove extracts, which inhibits the biosynthesis of ergosterol, a component of the microbial cell membranes. These may lead to the disruption of microbial cell membrane permeability causing cell death . Other researchers have attributed the antifungal activity to eugenyl acetate which inhibits germ tube formation, prevents the formation of candidal biofilms, and enhances phagocytic activity of macrophages against the C. albicans species . The current study revealed that S. aromaticum extracts exhibited strong antifungal potency against Candida species at low concentrations (250 μg/disc). Hence, it could be used as a potential source of natural antifungal drugs. The cytotoxicity assay confirmed that the diethyl ether clove extract possessed the lowest toxicity, while the ethyl acetate extract exhibited moderate toxicity against the HUH7 cancer cell lines, with IC 50 values of 62.43 and 33.68 μg/ml respectively. These results were in accordance with that of Kumar et al. who have reported clove oil toxicity against MCF-7 human breast cancer cell lines, with an IC 50 value of 36.43 μg/ml . Vijayasteltar et al. have demonstrated the safety of clove bud extracts as dietary supplements in Wistar rats, confirming the absence of toxicological changes in behavioral observations, body weights, organ weights, ophthalmic examinations, feed consumption, hematology, urinalysis, and clinical biochemistry parameters compared to the untreated group of animals .
Conclusion Syzygium aromaticum extracts exhibited a highly antifungal efficiency against the most common and predominant strains causing candidal vaginitis. Ethyl acetate was the most effective organic solvent in the extraction process, producing a high yield of clove active constituents. Furthermore, the results demonstrated a high potency of the clove extract compared with terbinafine (control). Hence, it could be used as a natural, safe, and effective antifungal agents. Moreover, it could be prescribed as a substitute to several chemotherapeutic agents used in the candidal vaginitis therapy for external use, eliminating the extensive side effects associated with these chemical agents, especially in pregnant women. Fig. 1 1 Fig. 1 Minimum inhibitory concentration of S. aromaticum ethyl acetate extract against Candida species
Fig. 2 2 Fig. 2 Chromatogram of S. aromaticum ethyl acetate extract
Fig. 3 3 Fig. 3 Chromatogram of S. aromaticum diethyl ether extract
Fig. 4 4 Fig. 4 Chromatogram of S. aromaticum methanolic extract
Fig. 5 5 Fig. 5 Chromatogram of S. aromaticum n-hexane extract
Table 1 1 Antimicrobial activity of different S. aromaticum extracts against Candida strains S. aromaticum extracts Inhibition zone diameter (mm) of (10 mg/ disc) Candida strains C. albicans C. glabrata C. tropicalis Diethyl ether extract 19.43 ± 0.15 15.93 ± 0.15 23.70 ± 0.17 Ethyl acetate extract 20.93 ± 0.32 14.90 ± 0.21 30.77 ± 0.95 Methanolic extract 16.03 ± 0.83 12.93 ± 0.20 25.60 ± 1.16 N-hexane extract 18.93 ± 0.03 14.07 ± 0.09 30.37 ± 0.26 Control (50 μg/disc) 21.50 ± 0.06 9.60 ± 0.06 24.33 ± 0.13 Eugenol 15.67 ± 0.34 11.21 ± 0.24 19.93 ± 0.42
Table 2 2 Minimum inhibitory concentrations of S. aromaticum ethyl acetate extract against pathogenic Candida strains Concentration of Inhibition zone diameter (mm) of Candida strains extract (mg/disc) C. albicans C. glabrata C. tropicalis 0.125 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.250 0.0 ± 0.0 0.0 ± 0.0 8.40 ± 0.06 0.500 8.83 ± 0.16 8.07 ± 0.09 13.40 ± 0.17 1.000 13.77 ± 0.72 11.30 ± 0.12 20.73 ± 0.72 2.000 15.73 ± 0.03 12.60 ± 0.06 23.10 ± 0.17 4.000 16.83 ± 0.32 13.83 ± 0.38 24.17 ± 0.20
Table 3 3 Phytochemical components of S. aromaticum ethyl acetate extract Compounds Chemical M.W. Retention % of formula time (min.) Total Eugenol C 10 H 12 O 2 164 16.33 58.88 trans-Caryophyllene C 15 H 24 204 17.41 14.44 α-Humulene C 15 H 24 204 18.31 1.88 Eugenol acetate C 12 H 14 O 3 206 20.27 23.86 Longipinocarvone C 15 H 22 O 218 21.30 0.19 Caryophyllene oxide C 15 H 24 O 220 21.45 0.74 M.W. Molecular weight diameter of 8.4 mm. The MIC against C. albicans and C. glabrata was 0.5 mg/disc, with inhibition zone diameters of 8.83 and 8.07 mm, respectively (Table 2 ). The MIC of S. aromaticum ethyl acetate extract against C. tropicalis was lower than that against both C. albicans and C. glabrata. Hence, C.
Table 4 4 Phytochemical components of S. aromaticum diethyl ether extract Compounds Chemical M.W. Retention % of formula time (min.) Total Eugenol C 10 H 12 O 2 164 16.21 66.48 trans-Caryophyllene C 15 H 24 204 17.38 1.63 α-Humulene C 15 H 24 204 18.29 0.21 Eugenol acetate C 12 H 14 O 3 206 20.21 31.37 Caryophyllene oxide C 15 H 24 O 220 21.44 0.31 M.W. Molecular weight
Table 5 5 Phytochemical components of S. aromaticum methanolic extract Compounds Chemical M.W. Retention % of formula time (min) total Chavicol C 9 H 10 O 134 14.09 0.78 α-Cubebene C 15 H 24 204 15.50 0.79 Eugenol C 10 H 12 O 2 164 16.44 55.58 trans-Caryophyllene C 15 H 24 204 17.46 15.71 α-Humulene C 15 H 24 204 18.32 2.46 α-Farnesene C 15 H 24 204 19.50 0.37 β-Cadinene C 15 H 24 204 19.84 0.59 Eugenol acetate C 12 H 14 O 3 206 20.33 19.83 Caryophyllene oxide C 15 H 24 O 220 21.47 1.10 Musk ketone C 14 H 18 N 2 O 5 294 29.52 2.78 M.W. Molecular weight compound, with relative percentages of 66.48, 58.88, 55.58, and 61.37%, respectively.
Table 6 6 Phytochemical components of S. aromaticum n-hexane extract Compounds Chemical M.W. Retention % of formula time (min.) Total Eugenol C 10 H 12 O 2 164 16.27 61.37 trans-Caryophyllene C 15 H 24 204 17.39 0.52 α-Humulene C 15 H 24 204 18.30 0.34 Eugenol acetate C 12 H 14 O 3 206 20.26 34.95 Caryophyllene oxide C 15 H 24 O 220 21.45 0.82 M.W. Molecular weight
|
10.1097/md.0000000000016945
|
cc-by|cc-by-nc
| null | null |
openalex
|
Background: Traditional Chinese medicine (TCM) has gradually drawn the attention of clinicians as an alternative choice for insomniacs and TCM yangxin anshen therapy (TYAT), as a crucial therapy of treating insomniacs, is based on the theory of syndrome differentiation. However, owing to the lack of evidence-based medical evidence, the authors intend to carry out this study to evaluate TYAT's effectiveness and safety. Methods: Seven electronic databases will be searched from inception to July 2019. Two authors will independently identify randomized controlled trials, fetch data and assess the risk of bias with tools provided by Cochrane. A comprehensive meta-analysis will be conducted with the Cochrane Collaboration software (Review Manager 5.3) for eligible and appropriate studies. Further, the evidence will be assessed with the grading of recommendations assessment, development, and evaluation approach. Results: This article will be dedicated to assessing TYAT's efficacy and safety for insomniacs.
Conclusion: This systematic review and meta-analysis may provide persuasive evidence for the clinical application of TYAT in treating insomnia.Introduction Insomnia refers to a subjective experience that feels dissatisfied with sleep duration or quality and affects daytime social functioning despite appropriate sleep opportunities and sleep environments. In industrialized countries, about 6% of adults are afflicted with chronic insomnia. A cross-border European study among the elderly in 16 European countries found that the prevalence of insomnia ranges from 16.6% in Denmark to 31.2% in Poland. It is estimated that the proportion of the general population suffer from insomnia in China is about 15%, which is on the low side compared with the western countries but is close to reports from Asian countries. At the same time, the threat posed by insomnia to human health has gradually gained attention. Studies have found that chronic insomnia increases the risk of cardiovascular disease, especially the reduction of objective sleep duration was found to be a risk factor for hypertension. [4, 5] According to a prospective study, both difficulty initiating sleep and nonrestorative sleep are associated with a modestly higher risk of mortality. Besides, insomnia also places a substantial socioeconomic burden. [7, 8] Currently, clinical medications for insomniacs in China mainly include benzodiazepine drugs, nonbenzodiazepine drugs, melatonin receptor agonists, orexin receptor antagonist, along with the antidepressant drugs with hypnotic effects. However, these drugs have limitations of one or more undesirable adverse effects, such as the dizziness, headache, forgetfulness, bitter mouth, fatigue, withdrawal reaction, lethargy, hangover, falls, and so forth. Looking back on the past over 2000 years of history, herbal medicines have been extensively used to treat insomniacs in China. According to the theory of traditional Chinese medicine (TCM), the heart ("Xin" in Chinese Pinyin is used in this paper to distinguish it from the anatomically described organ) is thought to be related to the pathogenesis of insomnia in line with its function of the "seat of consciousness" and the place where the spirit or mind ("Shen" in Chinese Pinyin) is located, which can guide the follow-up treatments. Based on this, "Xin" is considered to be closely related to the occurrence of insomnia and TCM yangxin anshen therapy (TYAT), which can be defined as the therapy of tranquilizing the mind by nourishing the heart, is one of the crucial therapeutic principles for insomnia in TCM. Previous meta-analyses [12, 13] have suggested that Chinese herbal medicine is a potential alternative therapy for insomnia. However, due to the heterogeneity of different studies, the evidence cannot conclusively confirm the feasibility of the clinical application of herbs in insomniacs. Given that the above metaanalysis did not take the differences of TCM therapeutic principles among studies into account, the authors plan to conduct this study to provide evidence-based medical evidence for the clinical application of TYAT for insomniacs.
Methods
Protocol register This protocol of systematic review and meta-analysis has been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols statement and has already been registered on the PROSPERO platform (https:// www.crd.york.ac.uk/PROSPERO/) with an assigned registration number CRD42019135115.
Ethics Since this is a protocol with no patient recruitment and personal information collection, no further ethical approval is required.
Database search strategy Computer retrieval and manual retrieval will be used to retrieve all the published literature independently by 2 authors. Databases searched include China National Knowledge Infrastructure, Chinese Scientific Journals Database, Wanfang Database, China Biological Medicine Database, PubMed, EMBASE Database and Cochrane Central Register of Controlled Trials. All randomized controlled trials (RCTs) on TYAT for insomnia will be collected from inception of each database to July 2019. The specific search strategy will be formulated with the specific database. Among them, the author lists the retrieval strategies of the PubMed database (see Table 1 ). It will also be supplemented by a manual search of relevant literature.
Eligibility criteria and elimination criteria 2.4.1. Types of participants. The included patients should be explicitly diagnosed with insomnia, regardless of gender, ethnic background, or nationality. All subjects must meet the corresponding criteria: diagnostic and statistical manual of mental disorders, international classification of diseases, Chinese classification and diagnostic criteria of mental disorders, or Chinese guideline for the diagnosis and treatment of insomnia in adults. Studies involving other diseases will be ruled out, such as severe mental illness, drug laziness, and so forth.
Types of interventions. Interventions assigned to the experimental groups must be an oral prescription that embodied the therapeutic principles of TYAT. The intervention in the control group could be benzodiazepines, nonbenzodiazepine hypnotics, placebo, or other basic treatment. The dose, dosage form, and treatment duration of the 2 groups will not be taken into considerations in this study. Studies involving acupuncture, moxibustion, massage, and other TCM prescription will be eliminated. Additionally, the authors are about to eliminate studies involving unfixed TCM prescriptions.
2.4.3. Types of outcome measures. The primary outcome measures adopted in this study are polysomnography and the Pittsburgh sleep quality index (PSQI). The secondary outcome measures are clinical efficiency and adverse events. It is supposed to be noted that the criteria for clinical efficiency are divided into TCM curative efficacy [19, 20] and PSQI curative efficiency in our study.
Type of study. RCTs that focused on TYAT for sleep disorders, whether or not blinding, will be included. Only Chinese and English literature will be taken into account. However, quasi-RCTs enrolled according to medical record number or birthday will not be set out in the present paper.
Study selection and data collection Two investigators are in charge of completing the literature search according to the search strategy, respectively. Retrieved articles will be included in the form of titles and abstracts for further screening. The reasons for literature rejection will be recorded separately. In case of disagreement between the 2 investigators, they should settle through negotiation first. If necessary, the third investigator ought to be invited for determination. The flow chart is presented in Figure 1 . Finally, 2 authors are expected to extract the data of included literature respectively to make a characteristic table, which includes the first author, year of publication, diagnostic criteria, sample size, interventions, the course of treatment, outcome measures, and follow-up time.
Literature quality assessment The quality evaluation of the RCTs to be included will be completed using the tools recommended by the Cochrane Collaboration. The judgment of bias risk includes 7 aspects: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. The score of each article is distributed from 0 to 7. Two investigators will conduct the quality evaluation separately and invite a third reviewer to deal with any disagreement.
Statistical analysis methods The Review Manager 5.3 software will be implemented for statistical analysis. Dichotomous data will be analyzed by using the risk ratio with 95% confidence interval (CI) and continuous variables will be analyzed by using the mean difference with 95% CI. For statistical heterogeneity, chi-square test and I 2 index for measurements are used. If there is no statistical heterogeneity among the studies (P > .10, I 2 < 50%), the fixed-effect model will be employed. If there is statistical heterogeneity among studies (P .10, I 2 >= 50%), the sources of heterogeneity will be analyzed, and subgroup analysis may be undertaken. If there is statistical heterogeneity but no clinical heterogeneity among studies, the random effect model will be employed for analysis. If heterogeneity obviously exists between studies, only the descriptive analysis will be promoted. Sensitivity analysis will be carried out by changing the effect model and statistical methods. Only when the subgroup included >10 studies, publication bias can be measured.
Grading of recommendations assessment, development, and evaluation quality assessment This assessment will be carried out through the Guideline Development Tool (https://gradepro.org/). On account of grading of recommendations assessment, development, and evaluation handbook, 2 independent authors will classify the quality of evidence into the following 4 levels: high quality, moderate quality, low quality, and very low quality.
Discussion TCM syndrome (TS), the basic characteristics and a core concept, refers to a unique diagnostic method for classifying different individual pathological conditions under the guidance of TCM theory, which can be regarded as subtypes of modern diseases. Differentiation of TS, a basic feature that runs through the development history of TCM for more than 3000 years, is the basis for all TCM doctors to carry out clinical diagnosis and treatment. Differentiation of TS mainly based on clinical features would further contribute to classifying patients to improve the curative efficacy of TCM-associated therapeutic measures. In a nutshell, each kind of TS corresponds to its own therapy and making therapeutic regime based on TS differentiation is the key to enhance clinical efficacy. As mentioned earlier, previous several meta-analyses [12, 13] did not take the differences of TCM therapies among studies into account. Therefore, there is a necessity to comprehensively retrieve and rigorously carry on high-quality research. To some extent, our appropriate protocol designed ahead may ensure that future evaluations of specific therapy for insomniacs like TYAT are more likely to provide objective evidence of guiding value. For all we know, this study will be the first attempt to evaluate the evidence of TYAT in the treatment of insomniacs. It has to be acknowledged that some restrictions may affect the drawn conclusion based on this protocol. Retrieved studies will only include those published in Chinese or English. If certain differences in the diagnose criteria, interventions, dosage, duration of medication, and so forth could be not properly addressed, excessive heterogeneity may result, which may affect the conclusion. Figure 1 . 1 Figure 1. Flowchart of study screening.
Table 1 1 PubMed search strategy draft. Sleep Initiation and Maintenance Disorders [MH] OR Disorders of Initiating and Maintaining Sleep [ALL] OR DIMS Disorders of Initiating and Maintaining Sleep [ALL] OR Early Awakening [ALL] OR Awakening, Early [ALL] OR Nonorganic Insomnia [ALL] OR Insomnia, Nonorganic [ALL] OR Primary Insomnia [ALL] OR Insomnia, Primary [ALL] OR Transient Insomnia [ALL] OR Insomnia, Transient [ALL] OR Rebound Insomnia [ALL] OR Insomnia, Rebound [ALL] OR Secondary Insomnia [ALL] OR Insomnia, Secondary [ALL] OR Sleep Initiation Dysfunction [ALL] OR Dysfunction, Sleep Initiation [ALL] OR Dysfunctions, Sleep Initiation [ALL] OR Sleep Initiation Dysfunctions [ALL] OR Sleeplessness [ALL] OR Insomnia Disorder [ALL] OR Insomnia Disorders [ALL] OR Insomnia [ALL] OR Number
|
10.1371/journal.pbio.1001145
|
cc-by
| null | null |
openalex
|
Primers provide a concise introduction into an important aspect of biology highlighted by a current PLoS Biology research article.Superantigens are bacterial proteins that generate a powerful immune response by binding to Major Histocompatibility Complex class II molecules on antigen-presenting cells and T cell receptors on T cells. A recent article reveals that at least one of the superantigens, staphylococcal enterotoxin B (SEB), also binds the co-stimulatory molecule CD28, suggesting that a much larger and potentially more stable complex is formed at the immunological synapse than was previously thought. This revelation greatly clarifies some of the mystery surrounding how and why these toxins are able to elicit such a toxic immune response at extremely low concentrations. These findings also highlight a novel role for CD28 in microbial pathogenicity. Bacterial superantigens (Sags) constitute a family of very stable bacterial proteins that are the most potent known activators of the immune system. They can cause food poisoning or, if they occur at sufficient concentration in the blood or lymphoid tissue, systemic shock . Those unfortunate enough to eat food contaminated with Staphylococcus aureus will experience a brief but violent episode of vomiting and diarrhoea just a few hours later-the gut's attempt to expel the Sag before it wreaks havoc with the immune system. If a Sag does get into the bloodstream, and if the patient has no neutralising antibody from previous exposure, then the Sag will induce a sudden and profound T cell stimulation that generates a cascade of cytokines, resulting in symptoms that include high fever, headache, vomiting, hypotension, aches, and rash, causing the condition known as Toxic Shock Syndrome. This life-threatening illness is often associated with young females who have developed an intra-vaginal infection of a staphylococcal strain producing the Sag Toxic Shock Syndrome Toxin (TSST) [2, 3] . Deep tissue infections by Streptococcus pyogenes can also produce similarly powerful Sags capable of causing lethal shock . Interestingly, the Saginduced immune response is not targeted at the bacteria themselves, but rather Sags function to direct a nonspecific T cell-and cytokine-mediated immune response that somehow assists in bacterial survival. Although many cytokines are produced in response to a single Sag, acute toxicity is blamed on the excessive production of three T cell cytokines-Interleukin-2 (IL-2), Interferon-c (INF-c), and particularly Tumour necrosis Factor a (TNF-a) [4, 5] . Perhaps the most notable feature of Sags is their extreme potency: many of the more than 30 different staphylococcal and streptococcal Sags stimulate profound proliferation and cytokine production in up to 20% of all peripheral T cells, at concentrations in culture that approach 1 femtomolar (10 215 moles/l). This is especially remarkable since T cells are not directly involved in the immediate defence against these bacteria. Why S. aureus and S. pyogenes should produce such a powerful T cell response has never been clearly resolved. One hypothesis proposes that Sags are important in the very early stages of colonisation when the bacteria are struggling to establish a niche. By stimulating local T cells, Sags may suppress the recruitment and activation of their real enemy-neutrophils and macrophages, which would destroy the bacteria. Sags must therefore be effective at vanishingly low concentrations and it is only when the bacterial colony becomes well established and fails to shut off Sag production that the toxic sequelae arise-a state that can be of little benefit to the bacteria and even less benefit to the host. Normally, microbial antigens are internalized by antigen presenting cells, digested, and then presented as small peptides on the cell surface bound together with Major Histocompatability Complex class II (MHC class II) molecules; the combination of MHC and peptide is then recognized by T cell Receptors (TcRs) expressed on T cells, thus stimulating an immune response specific to that peptide antigen. However, over the past two decades many elegant studies have revealed that all Sags do one thing very well-they hijack T cell antigen recognition by directly cross-linking MHC class II and TcR, thus bypassing the antigenpresenting stage and stimulating a much larger, inappropriate immune response. All Sags therefore have at least two separate binding sites-one for MHC class II and another for the b-chain of TcR . Mutagenesis studies have mapped these sites for a number of different Sags and co-crystal structures of Sags bound to either MHC class II or TcR have confirmed their location [7, 8] . What has been most surprising is the variety of binding modes used by individual Sags (Figure 1 Staphylococcal enterotoxin H (SEH) is the only Sag that binds to a TcR a-chain (Figure 1 ) . Thus although Sags share a very similar protein structure, each has evolved its own way of binding to MHC and TcR-and this remarkable binding diversity clearly offers S. aureus and S. pyogenes an important survival advantage. Although this Sag-MHC-TcR trimer model of Sag activation is universally accepted , there are several perplexing aspects of Sag behaviour that have never quite gelled, hinting that something else might better explain their extreme potency and toxicity. For example, the binding affinities that Sags display towards TcR and MHC class II are typically quite weak-in the low micromolar range, yet Sags stimulate at concentrations several million times lower than this . Sags are also several orders of magnitude less potent towards mouse T cells than human T cells, even when presented on human MHC class II -a discrepancy that has never been fully explained. Mutating the TcR binding site on Sags creates mutants that have no mitogenicity yet still retain significant toxicity especially when combined with small amounts of the Gram negative bacterial pyrogen Lipopolysaccharide (LPS). This is a particularly lethal combination, suggesting that mitogenicity and toxicity may utilize separate mechanisms . Finally, full activation and cytokine production demands that T cells receive a second costimulatory signal through the CD28 molecule, yet there was no evidence that CD28 was effectively engaged during Sag activation. Even so, T cells defective in CD28 are much less responsive to Sag and CD28 deficient mice are completely resistant to Sag toxicity, producing no TNF-a or INF-c [11, 12] . It is well-known that CD28 on its own can deliver a potent signal to T cells. This is best highlighted by the unfortunate 2006 clinical trial of the company TeGenero's humanised anti-CD28 monoclonal antibody TGN1412: unexpectedly all six healthy volunteers suffered near fatal toxic shock immediately following Figure 1 . The model structures of four trimer complexes reveal the diversity in Sags binding. In the first structure, SEC3 (similar to SEB) is bound to the conserved MHC class II a-chain and engages a Vb8 domain [18, 19] . Note how the TcR makes no contact with the normal peptide/MHC surface as it would during normal peptide recognition. In the second structure TSST binds in the same location as SEC3 but in a different orientation. TSST is very specific for the human Vb2 TcR [20, 21] . In the third structure, the streptococcal Sag SPEC is bound to the other side of MHC class II and ligates human Vb2 TcR [8, 22] but in a quite different orientation to TSST. The fourth structure is of staphylococcal enterotoxin H (SEH) bound to the bchain of MHC class II but ligating a TcR through its Va27 domain-the only Sag known to engage a TcR a-chain . In each of the structures the location of the CD28 binding identified by Arad et al. is represented by red space filling spheres. Note its position well away from both TcR and MHC class II in a suitable position to engage a CD28 molecule in a tetrameric membrane complex in each of the four Sag orientations. doi:10.1371/journal.pbio.1001145.g001 injection of a small amount of TGN1412 . In an insightful letter in the New England Journal of Medicine discussing this unfortunate incident, David Corry and Dorothy Lewis from the Baylor College of Medicine proposed a linkage between the anti-CD28 toxicity observed in this trial and Sags: '' together these observations indicate that T cells activated by superantigen binding to both CD28 and antigen receptor mediate the toxic shock syndrome'' [14, 15] . New evidence, presented in this issue of PLoS Biology, suggests they were right! A paper in this issue of PLoS Biology has carefully reexamined the Sag staphylococcal enterotoxin B (SEB) and follows on from an earlier study that first identified peptide antagonists of SEB toxicity . SEB is best known for its food poisoning abilities, and although it is a relatively weak human T cell mitogen compared to other Sags, it produces large amounts of IL-2, TNF-a, and INF-c when added to human peripheral blood cells and kills mice that have been pre-sensitised with the liver toxin D-galactosamine . In addition to MHC class II and TcR, Arad et al. found that SEB possesses a third binding site to-you guessed it-CD28. This is a revelation and in hindsight makes perfect sense, providing a much clearer rationale for the extreme potency and toxicity exhibited by SEB and possibly other Sags. These findings also highlight a novel role for CD28 in microbial pathogenicity. The authors show that SEB binds to a soluble form of CD28 through a relatively conserved region that is distinct from both the MHC class II binding site and the TcR binding site (shown by the red spheres in Figure 1 ) . They use short synthetic peptides that mimic both the SEB and CD28 regions to effectively block SEB-induced cytokine production by T cells. To strengthen their case, the authors mutate the predicted CD28 binding site on SEB and show that this mutant fails to stimulate IL-2, TNF-a, and INF-c in T cells. SEB is also shown to bind to cells transfected with CD28 that lack either MHC class II or TcR. Finally they use the technique of peptide phage display to generate novel synthetic peptides screened for inhibiting SEB binding to CD28-Fc. These peptides, as well as peptides derived directly from CD28, protect mice from SEB-induced lethality. Importantly the authors show that the same CD28 binding site can be found on other Sags such as SEA and TSST and that the synthetic peptides that inhibit SEB also inhibit SEA-and TSST-induced cytokine production . This suggests that CD28 is a general target for all bacterial Sags. On the opposing side of the complex, the SEB binding site on CD28 is mapped to a region needed for homo-dimerisation that is distinct from the B7 binding site. CD28 may therefore be bound by both SEB and B7 in the resulting membrane complex (Figure 2 ). While this discovery greatly clarifies the mechanism of Sag toxicity, there are some nagging questions arising from this study that will need further work. The linear region identified-SEB 150-161 -is predominantly a loop structure that connects the b8-strand to the first bit of the highly conserved a4 helix situated at the core of all staphylococcal and streptococcal Sags. The 14-mer region used in these studies-VQTNKKKVTAQELD-is reasonably conserved in other Sags and is located on a face distinct from the MHC class II and TcR binding sites (Figure 1 ). The problem is that the majority of amino acid side chains in this sequence are buried within the native SEB structure with only 4 of the 14 side-chains exposed to the solvent and thus available to contact CD28. At the moment it's difficult to see how a linear synthetic peptide of this region can block CD28 binding when the structure it competes with is not solvent accessible. Another curious finding is the functional mutant SEB-T150A.K152A, which fails to induce cytokines in T cells but actually binds to soluble CD28 with higher affinity than native SEB. This is the opposite of what would be expected but could be explained by a model where optimal signalling through CD28 requires only transient interaction with SEB. The authors have focused only on cytokine gene expression and protection from lethal toxic shock as the means of assessing SEB activation. It would have been nice to see whether T cell proliferation is also affected to the same extent as cytokine production. If it is not, then this would suggest that T cell proliferation is primarily mediated through TcR ligation while the excessive cytokine toxicity results from CD28 ligation. Like any revealing discovery, more questions are raised than answered-questions that will certainly be addressed in future studies. There is no doubt that a new model that places Sags in the middle of a large membrane complex consisting of MHC class II, TcR, and now CD28 will be extensively tested in the coming months. At this stage however, one can only marvel at the extraordinary efficiency of these small protein toxins to engage the three most crucial molecules in T cell antigen recognition. Exactly why they do this is the next question, but given this new CD28 connection, there are now new approaches for developing therapeutics against toxic shock. CD28 is essential for Sag toxicity [11, 12] . In the new model proposed on the right, CD28 is part of a larger more stable complex directly ligated by Sag. This new model explains much better the extreme potency and cytokine toxicity observed with Sags. In theory this tetrameric complex would be more stable being held together by 3 interactions rather than 2. doi:10.1371/journal.pbio.1001145.g002 ). For example Staphylococcal enterotoxin B (SEB) and C (SEC) cross-link MHC class II a-chain and TcR b-chain. Streptococcal pyrogenic exotoxin C (SPEC) binds to the other side of MHC class II and cross-links TcR b-chain while Staphylococcal Enterotoxin A (SEA) binds both aand b-chain binding sites on MHC class II to cross-link TcR b-chain.
Figure 2 . 2 Figure 2. A cartoon of what might be happening at the immunological synapse during Sag engagement. In a model consistent with the crystal complexes shown in Figure 1, CD28 provides the essential 2nd co-stimulatory signal through separate B7 ligation.CD28 is essential for Sag toxicity [11, 12] . In the new model proposed on the right, CD28 is part of a larger more stable complex directly ligated by Sag. This new model explains much better the extreme potency and cytokine toxicity observed with Sags. In theory this tetrameric complex would be more stable being held together by 3 interactions rather than 2. doi:10.1371/journal.pbio.1001145.g002
September 2011 | Volume 9 | Issue 9 | e1001145
PLoS Biology | www.plosbiology.org
|
10.57096/edunity.v2i9.150
|
cc-by-sa
| null | null |
openalex
|
Human Resource Development is any effort to improve the implementation of current and future work, by providing information, influencing attitudes, or adding skills, in other words, development is any activity intended to change behavior consisting of knowledge, skills, and attitudes. One of the objectives of human resource development according to Hasibuan is to improve services. The focus of the research conducted by the author is on the service of making birth certificates. The method used is a qualitative research method which is a method for exploring and understanding the meaning that a number of individuals or groups of people ascribe to problems. The informant retrieval technique uses Purposive Sampling which is a sampling technique with certain considerations. This technique includes people who are selected on the basis of certain criteria that are considered capable of providing information in accordance with the focus of the research. The results of this study are based on the dimensions of the research that in terms of knowledge it is still said to be not optimal due to formal education that does not meet the criteria and also lack of participation in non-formal education. For the skill dimension, it is also still not optimal because there is no special training on making birth certificates. However, the attitude and behavior dimensions are optimal. The factors that become obstacles are the rapid development of technology and the lack of discipline of employees. In addition, the efforts made are to carry out direction, supervision, and as well as sharing knowledge between fellow employeesIntroduction Human resources are productive individuals who act as the driving force of an organization, either institution or company (Susan, 2019) , The mission of human resources is to become an asset whose talents must be trained and developed. Therefore, an employee or employee is not just a resource, but capital or assets for an institution or company, which can be invested through development (Subyantoro & Suwarto, 2020 ). An organization can run well if there are human resources who act as its driving force. Here the importance of the role of leaders in human resource management in order to be able to move the organization in achieving goals. One of them is how does a leader improve the quality of his employees or employees by doing development (Supardi, 2016) . In a strong organization must have quality human resources. To manage these resources, organizations need to accelerate them, one of which is by developing human resources. This development is important because it is enabled to carry out the duties and responsibilities given by the organization (Riniwati, 2016) . Development of human resources is any activity intended to develop knowledge, skills, and attitudes, in order to help carry out work properly (Solong, 2020) . Investment in human resource development is an expenditure aimed at improving the productive capacity of employees so that they are able to face the demands of tasks, especially responding to the ever-changing demands of the future. In general, human resource development is a kind of activity carried out by companies or institutions to improve human resource skills (Suprihanto & Putri, 2021) . According to Rivai and Basri, (Sinambela & Sinambela, 2019 ) "that performance is the result or level of success of a person or whole during a certain period in carrying out tasks compared in advance and agreed upon". This means that the results of the performance of the apparatus depend on how superiors or agencies empower existing human resources during a certain period. Employee performance is also a benchmark for how successful leaders are in developing human resources (Susilowati & Farida, 2019) . The government has stipulated Law no. 5 of 2014 concerning the State Civil Apparatus was structured as a bureaucratic reform program which is a solution to changing the Indonesian government's bureaucratic order. In accordance with the management of the State Civil Apparatus, it must also change from personnel administration to the development of human capital. This is the policy and management of the State Civil Apparatus which is based on qualifications, competence, and performance that is fair and without tendencies and regulates issues of promotion, transfer, and even dismissal of the state civil servants. The Government Regulation Number 101 of 2000 concerning the Education and Training of Civil Servants, on this basis, it is necessary to build a state civil apparatus that has integrity, is professional, neutral, and free from political intervention, free from corruption, collusion and nepotism practices, and is able to organize public service for the community and able to carry out the role as an adhesive element for the unity and integrity of the nation (Wicaksana, 2014) . According to (Hasibuan & Hasibuan, 2021) one of the objectives of developing human resources is to improve service to the community. Service is basically an activity that provides benefits by one party to another but does not have a physical form and without any change in ownership (Hardiyansyah, 2018) . Service according to the Big Indonesian Dictionary has 3 meanings, namely: How how to serve, efforts to meet the needs of others by getting rewards, and the facilities provided in connection with the sale and purchase of goods or services. An organization, especially an organization that is directly related to the community, of course it willgive the best service. In order to realize the best service, competent employees are needed in carrying out their duties. This indicates the importance of developing human resources in order to improve the ability of employees so that they are able to provide the best service to the community. The Department of Population and Civil Registration of Kuningan Regency is a government agency that has the task of carrying out household affairs for the Regional Government and duties in the field of Population and Civil Registration. Office of the Department of Population andRecording Kuningan Regency Civil Servants has a vision, namely "Prime in Population and Civil Registration Services" meaning that it must be able to realize maximum service to the community in administrationpopulation and civil registration. So as to create effective and efficient services in the process of population and civil registration services. Therefore, to improve the desired quality of the apparatus, it is necessary to have a good human resource development process. The Department of Population and Civil Registration has several fields in charge of providing services, one of which is the Civil Registration Services Division. Civil Registration Service is an activity of service and development regarding the legal status of a person at a time which can be used as authentic evidence for the party concerned or a third party. The forms of civil registration services are: making birth certificates, death certificates, marriage certificates, and divorce certificates, and others. The focus of the research conducted by the author is regarding services for making birth certificates. In initial observations, here the authors found several problems, where employees oftendifficulty in carrying out some of his duties so that the service to the community, especially in the manufacture of birth certificates, is disrupted. As according to the author's observations of the problems thatfaced the field of Civil Registration Servicesis as follows : 1. The still low level of officer education causes there to be some officers who do not yet understand the rules and procedures for making a birth certificate. 2. There is still a lack of employee skills in handling the making of birth certificates seen from their accuracy and also the ability to use equipment so that service to the community is not optimal, The above problems are certainly often encountered by employees specifically in the field of Civil Registration Services, however, until now there has not been any improvement or increase in the ability of employees to complete problem the. Yet according to research earlier namely (Misran, 2021)
Research methods The method used is a qualitative method, using a descriptive research design. The research technique used is purposive sampling technique. Data research techniques used include observation, interviews, and documentation. As well as testing its validity using data triangulation techniques.
Result And Discussion
Research result Research results are assessments that contain a complete description and analysis of the data and its interpretation. Research resultis the process of properly organizing and grouping information about an activity based on facts through the efforts of the researcher's mind in processing and analyzing research objects or topics in a systematic and objective manner to solve a problem or test a hypothesis so thatformed a general principle or theory. In According to (Solong, 2020), knowledge is defined as something that is known (intelligence) and everything related to what has been learned so that it is able to support it in its work. Knowledge is related to education, education here means formal education and non-formal education followed by employees. From the knowledge dimension based on the existing parameters, it is still not optimal, this is because seen from the formal education it is still not fulfilled criteria, both level of education and educational background, in terms of non-formal activities, in this case Dukcapil learns that many employees do not participate in these activities, causing employees to still not understand the procedures for servicing certain birth certificates, which these employees have not been able to complete, so they require help other employees in solving it.
Skills According to (Solong, 2020) an understanding of the meaning of skills comes from the word "skilled" which means proficient in completing tasks, capable and agile, so that skills are defined as the ability to complete tasks. Skills are the ability of employees to carry out tasks/technical work using the available equipment. Skill parameter consists of 1). Training, 2). understand the main tasks and functions, 3). complete the work on time and, 4). Don't procrastinate either. From the Skills Dimension, based on the existing parameters, it is still said to be not optimal, this can be seen from training activities that have not been maximized, such as online service training, and also the absence of special training activities regarding services the creation of birth certificates which the official who finally has to practice himself. From the perspective of understanding the Basic Tasks and Functions, the officers already understand even though some need to learn more because they have just been transferred. For punctuality in completing the task is also quite good even though it is a bit late even though the officer still when there is a given task is immediately done without delay.
Attitude/Behavior According to (Solong, 2020) Attitude is the regularity of one's feelings, thoughts and predisposition to act according to environmental aspects. The parameters of attitude/behavior include: 1). loyalty to the leadership, 2). innovative and high creativity, 3). love work, 4). like to work together, 5). very communicative towards the implementation of work assignments. From the Attitude/Behavior Dimension based on the existing parameters it can be said to be good/optimal, this can be seen from employee loyalty to every leader's directives, employees also try to develop creativity and innovation through the latest services to help the community, this is supported by a sense of belonging andlove employees towards their work and are also able to work well together, besides that employees are also communicative in their work.
Factors Inhibiting Human Resource Development in Birth Certificate Making Services One of the inhibiting factors is the rapid development of technology which causes some employees to have difficulty adaptingtoo late services, especially services for making application birth certificatesSNAP. Sofactor furthermoreis lessthe discipline employees in participating in human resource development, where the Kuningan Regency Population and Civil Registration Office has provided development activities in the form of Dukcapil Learning but employees rarely participate in these activities (Rosnaeni, 2021) .
Efforts That Have Been Made In Developing Human Resources In Birth Certificate Making Services Efforts have been made by the Department of Population and Civil Registration in the Development of Human Resources through direct supervision by the Head of Civil Registration in order to minimize the mistakes of employees in the service of making birth certificates. Apart from that, I gave directions so that the employees were not confused and knew what I needed to do. And lastis sharing knowledge between employees who already understand to employees who still don't understand (Amalia, 2020) . In sectionfurthermore the writer also found the results of research on the deedBirth. The definition of a birth certificate in terms of legal essence is one of the concrete forms of human rights, apart from that it is part of the population administration in which the state recognizes the personal status or status of a person on the registration of his birth based on law.positive (Ningrum, 2022) . primary lawRegarding birth certificates, it is regulated in: 1). Birth certificates are one of the services provided by the Kuningan Population and Civil Registration Service to the public, which is this task charged to the Service Sector. The flow and mechanism of self-made birth certificate services are as follows (Gunawan Nabilah Suci, 2023): 1. The applicant/community directly goes to the Kuningan Population and Civil Registration Office while bringing the required documents for making a birth certificate 2. Then the applicant takes the queue number while waiting until the queue number is called, after being called by the applicant/community immediately submits the file to the employee in charge of the service department. 3. The service officer will receive the file while checking the completeness of the file, if the file is incomplete then the service officer will return the file to be completed again. 4. If the file for making a birth certificate is correct, the officer will input the data and print a quote from the birth certificate. 5. The birth certificate extract that has been printed will be checked by the officer in charge of verification to be checked again, if there is an error then the birth certificate extract will be returned to the officer to be corrected. 6. After the quotation of the birth certificate passes the check, it will be submitted for signature by the Head of Service, then after receiving the signature, the quotation of the Birth Certificate is returned to the product delivery officer to be handed over to the applicant/community. In the Birth Certificate it is subdivided into 4 types based on the status and condition of the parents, namely: 1. A Father's Son.
Discussion
Development of Human Resources in Services for Making Birth Certificates at the Office of Population and Civil Registration Human resource development is an effort to improve human capabilities, especially in an organization to make it better. One of the goals of developing human resources is how to provide the best service to the community (Hasibuan. 2021: 70) . This also applies to government agencies such as the Population and Civil Registration Office of Kuningan Regency as service providers, especially services for making birth certificates, so quality employees are needed through human resource development. The quality of human resources has an important role in improving service. In realizing quality service employees must have a strategy that is preparing themselves to become servants for the whole community. In conducting research, here the author uses one of the theories of Human Resource Development according to Solong as a research analysis knife. (Solong, 2020) defines the notion of Human Resource Development as: "Every effort to improve current and future work performance, by providing information, influencing attitudes, or increasing skills, in other words, development is any activity intended to change behavior consisting of knowledge, skills, and attitudes." From the understanding above, the theory of human resource development according to (Solong, 2020) confirms that there are several indicators that need to be looked at, namely: 1. Knowledge 2. Skills 3. Attitude/Behavior In order to find out how human resource development has been carried out by the Population and Civil Registration Service in the service of making birth certificates, the author will conduct interviews with several informants, both key informants and supporting informants, using the 3 dimensions proposed by Solong, namely: knowledge, skills, and attitude/behaviour.
Knowledge Dimension According to (Solong, 2020) knowledge is defined as something that is known (intelligence) and everything related to what has been learned so that it is able to support it in its work. Knowledge is related to education, education here means formal education and non-formal education followed by employees. Knowledge Development is divided into 2 Parameters, namely: 1. Formal education 2. Non-formal education
Formal education Formal education, namely employees assigned by the Office to attend education carried out by educational institutions that are official. Education is a process of activities carried out to increase general knowledge or theories to employees in an organization which usually lasts a long time such as sending employees to a higher education level to provide increased work skills, theoretical and conceptual skills of employees, in this case means employees of the Field Civil Registration Service that serves the making of birth certificates. Based on the results of the author's interview with Mr. Nono Sumartono, Sos as the Head of the Civil Registration Services Division who is the main informant that: "As for standards, we actually haven't met those standards yet, but according to the work experience criteria, we are already qualified for the present, for the disciplinary criteria, we haven't because no one has yet gone to the basic science program of civil registration or knowledge that has lessons in public service. . Yes, we are to meet the standard of knowledge, the important thing is that he has high dedication, then he has loyalty to his work and he is capable of his work, we will appoint him. But if he is required to be academically, that is related to the risk of his own development, whether he wants to go back to school or not to take part in government programs funded by the government." (Interview on 8 May 2023 at 09.43 WIB) Based on the results of the author's interview with Mrs. Teti Tresnawati, SE Head of the Birth and Death Section, and Administrator of the Population Data Base who are supporting informants that: "Hmmm, in your opinion, I think it's ok, but if you mean Aa, maybe the education level should be at S1, there are indeed some who haven't S1, but they still have to be able to carry out their development even though their education is still not a bachelor's degree. Also, in terms of scientific discipline, yes, you are also not from a scientific discipline concerning government or service." (Interview on 11 May 2023 at 12.00 WIB) Based on the results of the author's interview with Mrs. Maryatiah as the Administration of Birth & Death Certificates where as a supporting informant that: "Hmm, in my opinion, judging from the employee education department, on average, there are some who have not graduated, one of them is like me, and also because my knowledge is still lacking, there are indeed a few obstacles in doing work, especially myself, who is in charge of administration. Birth certificate. Then there is no support for continuing education yet, the hope is that there will be support from the department, especially for employees who are still not up to standard." (Interview on 15 May 2023 at 14.00 WIB) Based on the results of the author's interview with Mrs. Tita Novita as an employee of the Administration of Birth & Death Certificates who is a supporting informant that: "I really want A to provide assistance from the Office for employees to continue their education, if there is, I want to continue with school A, but how about that? yeah A what about me myself from the Department." (Interview on May 22, 2023 at 12.34 WIB)
Data Validity From the data obtained from interviews with several informants and also supported by the observations made by the author, it can be stated ABSAH that employees have attended formal education, it's just that there are some employees who can indeed be said to have not met the criteria for both their level of education and also their scientific discipline is different from their job namely the service of making birth certificates.
Data analysis Based on the results of observations and interviews conducted by the author with several informants, it can be concluded that related to the formal education possessed by employees of the Civil Registration Sector. In providing services for making certificates, they do not meet this standard. education of several employees who carry out civil registration services that do not meet standards. The standard here means that at least employees can take college education or above. Unfortunately, in improving formal education, there is no real support from the Department of Population and Civil Registration, even so, employees inevitably have to be able to carry out the development themselves because it is already a risk to their jobs.
Conclusion Efforts have been made by the Department of Population and Civil Registration in the Development of Human Resources through direct supervision by the Head of Civil Registration in order to minimize the mistakes of employees in the service of making birth certificates. Apart from that, I gave directions so that the employees were not confused and knew what I needed to do. And the last is sharing knowledge between employees who already understand to employees who still don't understand. 2. Couple's Children from Husband and Wife 3. A Mother's Son. 4. The child's origin is unknown.
Figure 1 Types of birth certificate (Source: 2023 Research Results)
Development of Human Resources in Services for Making Birth Certificates at the Department of Population and Civil Registration of Kuningan Regency." Research Method Object of research in a study entitled "Analysis of Human Resource Development on Service Quality at the Office of the Population and Civil Registration Office of Enrekang Regency." Stating that the development of human resources is able to improve services to the community. Thus, on this basis it is deemed necessary to conduct research with the title " This research took place at the Department of Population and Civil Registration, located on Jl. RE. Martadinata No. 256, RW. 02, Ancaran, Kec. Kuningan, Kuningan Regency, West Java 45513.
Development of Human Resources in Services for Making Birth Certificates During this chapter the researcher explains the results of the study entitled "Development of Human Resources in Services for Making Birth Certificates at the Office of Population and Civil Registration of Kuningan Regency". The results of this study will be poured into several different sections so as to obtain specific research results regarding the correlation of research dimensions with problems in the field. As for the first part is: 1. How is Human Resource Development in the service of making Birth Certificates at the Kuningan Regency Population and Civil Registration Service? 2. What factors impede Human Resource Development in the service of making birth certificates at the Kuningan Regency Population and Civil Registration Service? 3. What efforts have been made by the Kuningan Regency Population and Civil Registry Office in carrying out Human Resource Developmentspecifically in the service of making Birth Certificates? This study uses a qualitative method in which the data obtained comes from interviews and observations with informants, as well as existing documentation if necessary. The interviews were conducted with informants who knew about the problems under study. The informants consist ofreport the key is the Head of Civil Registration Services, as well asreport supporters consisting of Civil Registration Service Officers who handle services for making birth certificates andpublic general. The informants needed in this study are: the observations and interviews, the results of the research obtained from the title "Development of Human Resources in Services for Making Birth Certificates at the Population and Civil Registration Office of Kuningan Regency". By using the indicators put forward by Solong regarding Human Resource Development as an analysis are as follows: 1. Knowledge Table 1. List of Key Informants and Supporters KEY INFORMANTS No Informant Name Department 1 Nono Sumartono, Sos Head of Civil Registration Service Division SUPPORTING INFORMANTS No Informant Name Department 1 Teti Tresnawati, SE Head of Birth and Death Section, 2 Maryatiah Administration of Birth & Death Certificates 3 Tita Novita Administration of Birth & Death Certificates 4 Dani General public 5 One General public (Source: 2023 Research Results)
Law Number: 23 of 2003 Concerning the Protection of Children and Women, 2). the law Number 35 of 2014 concerning Amendments to Law Number 23 of 2002 concerning Child Protection, 3). Law Number: 23 of 2006 concerning Population Administration, 4). Law Number: I4 of 2013 Concerning the First Amendment to Law Number: 23 of 2006.
[Human Resource Development in Birth Certificate Making Services at The Service of Population and Civil Registration of Kuningan District] Vol 2, No. 8, 2023
https://edunity.publikasikupublisher.com 955
Yanto Heryanto 1 *, Agus Dimyati 2 , Wendy Rivansah 3
|
10.11591/ijeecs.v17.i3.pp1383-1389
|
cc-by
| null | null |
openalex
|
In this work; we present an enhancement in blue laser diodes with new factors and applications for modern technology such as underwater telecommunications, bio-sensor and bio-medical systems etc. Years of advance meanwhile have much enhanced laser performance, and extremely improved their diversity, making lasers significant parts in scientific research, telecommunications, engineering, bio-medical imaging, materials working, and a swarm of other applications. This article viewing how laser technology has progressed to chance application requirements. The enhanced blue laser building diagrams to get a peak efficiency % at room temperature with modification. Moreover, we have as well estimated electro-optical performance packing of blue laser diodes been significantly various associated to GaAs laser method and novel developments and performances are required to enhance the optical power from anther laser diodes. Researchers need enhanced approaches to accurately make new the blue laser applications to use control of modern experimental measurements and optical communication.INTRODUCTION Blue beams are formed by gas lasers using He-Cad at 441.6 nm, and Argon-ion at 458-488 nm. Semiconductor with blue rays are naturally depending on gallium nitride. Both blue and violet lasers can also be built by means of frequency repetition of IR laser λ from DL or DPL . In the early 1990s the Institute of High Pressure Physics of Dr. Sylwester established knowledge to make gallium nitride quartzes with high organizational excellence . For example, diode lasers are semiconductor devices, they may as well be divided as semiconductor lasers .
METHOD AND RATE EQUATIONS OF THE MODELING BLUE LASER A laser diode is formed like a plane-paralleled rectangle where the two faces, semiconductors see, form a Fabry-Pérot resonator. This resonator is the source of the emission moved by distinguishing light emission as shown in Figure 1 . From Figure 2 & 3 shows energy level of DH-OS Laser Diode and the circuit of blue laser diode. To get a continua land powerful laser of the junction of semiconductors .
NUMERICAL SIMULATION (RATE EQUATIONS OF THE MODELING BLUE LASER) The energy levels in the conduction band and valence band of Quantum well are strongminded by resolving the 1D time independent Schrödinger equation. It's a measured typical which defines the performance of classification as a function of 3-D and time coordinates in standings of a wave function (Ψ). Solving the Schrödinger to get the probable Ψ that can occur within the section . - ħ 2 2m d 2 ψ(x) d 2 x + v(x)ψ(x) = Eψ(x) (1) substituting, ψ(x) = Asinkx + Bcoskx (2) ħ 2 k 2 2m (Asinkx + Bcoskx) (3) this gives E = ħ 2 k 2 2m (4) Outdoor the well, (x) = 0 and (x) = A sinkx. Hitting x = a, wherever (a) is the width of quantum: - kx = nπ (5) accordingly, the equivalence develops, E = ħ 2 π 2 n 2 2ma 2 (6) this calculation bounces the energy levels for changed values of n can be designed as Figure 4 : The Ψ can be plotted by giving, A = √ 2 a ( 7 ) which gives the equation ψ(x) = √2/a sin ( πnx a ) (8) Blue laser diodes produce light in the λ from 440 to 485 nm. This area of procedure has controlled to submissions in optical information loading and high-determination printing [14, 15] . AIN, GaN, InN materials all have a direct band gap.i.e. the optical transition across the band gab are allowed and therefore much strong than in the case of indirect band gaps as shown in Figure 5 , for example of SiC . Figure 5 . Shows band gap for different materials Resonant doubling arrangements permit higher efficiencies; 650mW in 430nm has been confirmed [20, 21] . Blue or near UV nitride-based semiconductor laser diodes (LDs) are nowadays commercially available. With regard to their short wavelength, lifetime and low power consumption they became suitable for submissions such as fast laser printing, high density optical information storage and, recently, in pico-size projectors occupied with lasers (400 -415) nm with a design centered at 408nm per the laser manufacturers' specification, this strategy was improved with a very large and forgiving clear aperture and is as well rewarded for the laser manufacturers' variation thickness . Schematic structure of Nakamura is shown in Figure 6 .
The Device Structure The laser diode assembly on a classical substrate as shown in Figure 7 , waveguide lasers were invented. Windows associated with the waveguides were imprinted into the SiO2 to contact the p-electrode. An Au bond pad was disappeared on top of the SiO2, contacting the p-electrode [24, 25] .
State of the Art Performance Level of the Device: The augmentation takes place in the junction of the semiconductor so only a small part of a small device is involved in the lasing processes. It banquets much less in the plane of the junction than in the plane vertical to the junction. This income that the beam is not TEM0,0, the divergence angles are relatively large, on the order of an insufficient degrees, and the divergence angles are changed in the various planes . Holes must be measured in the blue laser courses as well as electrons. The active method to variety an optical cavity for producing F.B is to usage reflected surfaces of planes slashed on the crystal . Figure 8 . Display the absorption of the key blue laser performance.
The Factor that Limit Performance The blue laser diode is a relatively recent innovation and did not enter commercial use until 2001. This was due to the difficulty of producing a suitable gain medium and the advances in materials science needed before the technology could become viable. It was primarily the product of research carried out in Poland and Japan, although the blue laser work confirmed the technical problems: -1. Nonexistence of an appropriate substrate.
the disappointment of p-type doping Scientists at Nichia determined the problematic of the discontinuity in lattice structure at the border molded. Future they enhanced upon the technique by using a buffer layer which is designed by deposit of GaN. The incapacitating was mixed, from Zn to Mg. The secondary blue laser diodes are twenty hundred periods brighter than the former forms .
Low Noise Low noise outcomes from the cavity construction which restricts the number of oscillation methods and maintains exact control of the component temperature. Only high-quality optical mechanisms are used, resulting in a noise specification of < 5.0% rms over a wide operating temperature range . A public portion of the amplitude noise is the comparative intensity noise : RIN = 〈ΔP 2 〉 〈P 2 〉≈[2×∆fS P (f)] 2 /〈P 2 〉 (9) where ∆f is the detection BW, SP(f) is the spectral density of the power fluctuations, and <P> is the mean P. RIN will reduction with growing pumping rate. If the laser is worked in a multi-mode regime [36, 37] . Specifications of the blue laser and values of parameters can be seen in Table 1 .
CONCLUSIONS Blue laser could be advantageous for communication determinations and taking the buildup the advancing. Modules of blue laser can today be designed to fit a broad range of applications [38, 39] . Depending on the required output power, amplitude stability, reliability, frequency tunability, compactness and output wavelength, one can choose between a wide set of laser technology. This study contributes with several new laser setups which can potentially replace much more complex and expensive systems or even enable the realization of new applications. This was possible due to the development of novel laser schemes, mostly utilizing nonlinear optics. The influential beam of blue laser has developed a significant instrument for spectroscopic analysis. A blue laser system is used for stimulating emission from solid models for spectrographic analysis. Additional quantities have been carried out utilizing like blue laser diodes for underwater data transmission and laser tracing. Researchers need enhanced approaches to accurately make new the blue laser applications to use modern experimental measurements. Figure 1 .Figure 3 . 13 Figure 1. Schematic of DH-OS laser diode
Figure 4 . 4 Figure 4. Ψ in quantum well
Figure 6 . 6 Figure6. Schematic structure of Nakamura, s blue laser
Figure 7 . 7 Figure 7. Construction of the InGaN multiple laser diode
Figure 8 . 8 Figure 8. Absorption of the main blue laser performance
ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci, Vol. 17, No. 3, March 2020 : 1383 -1389 1388
Table 1 . 1 Specifications of the Blue Laser and Values of Parameters Parameters Specifications Power 100,250 &350 mW λ 473nm Beam Size 1.4±0.2 mm Spacial Mode TEM00 BW <=40GHz Divergence <0.6 m rad Beam Angle 1 m rad Operation Temp. 15-35C 0
|
10.3390/foods9020112
|
cc-by
| null | null |
openalex
|
While the ethnic food market has become increasingly important in Italy, the effects of the hybridization of consumption patterns have been slowed by a consolidated culinary tradition. This study investigates the relationships among ethnic food consumption, food neophobia, and openness to different cultures with sociodemographic characteristics. A sample of 1317 Italian consumers responded to an online survey. The sociodemographic profile of the neophobic consumer appears to substantially differ from that of the consumer with an attitude of openness. Neophobic respondents are males, are older than 55 years of age, are less educated, have children, are retired, have difficulty meeting their financial responsibilities, and do not eat ethnic food. Respondents who are more open to different cultures are young adults, are highly educated, have no children, are employed, and are consumers of ethnic food. The relationship between food neophobia and openness to different cultures is confirmed to be the relationship between these variables and ethnic food consumption. The measurement of these characteristics could serve as a crucial indicator for analyzing the willingness to accept elements of novelty in an increasingly multicultural society. Additionally, consumers with the neophobic trait and who are less open to different cultures might have a less varied diet that is essential to good health.Introduction Italy has a consolidated culinary tradition that has its roots in regional cuisine. This tradition may serve as a stabilizing factor in the eating habits of Italians that slows the globalization of consumption patterns [1, 2] . However, Italy appears to be evolving with regard to food consumption. In recent decades, significant changes have occurred in the lifestyles and dietary habits of the Italian population, including an increase in dining out , the widespread presence of take-out and street food, a recreational attitude towards food particularly in young consumers , and increasing consumer demand in terms of food variety. Food polytheism is a term used to describe the current context of food consumption in Italy. Food polytheism refers to a subjective combination of food choices and highly complex and occasionally seemingly contradictory places of purchase and consumption [3, .
Increasing of Ethnic Food Presence in Italy The hybridization of food models is favored by different factors, including the globalization of markets and the mixture of populations through migration and the development of tourist exchanges with foreign countries . In recent decades, many social, political, and economic changes have fostered the arrival of significant flows of new residents into Italy who have promoted the development of economic activities in the food and catering sectors that are strongly linked to their countries of origin. The number of foreign entrepreneurs working in the catering sector increased by 43.3% from 2010 to 2014, leading to a record number of 21,176 owners of foreign enterprises (12.9% of the total number) . Therefore, ethnic foods are increasingly available on the Italian food market: Since 2007, ethnic food sales have increased by 93% . In addition to introducing new flavors and ingredients, ethnic food has cultural significance because it represents the food cultures and traditions of populations from other countries whose presence in Italy is increasing. For these reasons, foods from the culinary traditions of distant countries have started to have an increasingly important presence in Italy. However, their success has been delayed relative to what has been observed in other Western countries and has been less overwhelming than what has been witnessed in the Anglo-Saxon countries .
Consumption of Ethnic Food: The Role of Food Neophobia and Openness to Different Cultures Different interrelated aspects contribute to delineate consumption decisions, particularly regarding unfamiliar foods. They can be contextual (e.g., cultural or social), related to individual cognition/perception/sensation (e.g., the fear of negative consequences of eating a particular food or finding a certain type of food disgusting), or related to individual traits [12, 13] . The willingness to try non-traditional ethnic foods seems to be significantly predicted by a trait known as food neophobia [14, 15] , which is defined as a "reluctance to eat and/or avoidance of novel foods" . Several instruments have been developed to measure food neophobia as a behavior involving the rejection of foods in a particular situation and as a personal trait related to the propensity to avoid novel foods that remains constant over time and across different contexts . The Food Neophobia Scale (FNS) developed by Pliner and Hobden is considered a reliable and valid instrument for measuring neophobia as a personality characteristic in adults . The study of this psychological trait is important because it can affect the acceptance of new foods and influence consumers' daily food choices [19, 20] . Studies on children and adults suggest that food neophobia can affect the consumption of healthy foods (i.e., fruit and vegetables) [21, 22] and the willingness to try healthy alternatives (e.g., meat substitutes) . Additionally, neophilic individuals consume a broader variety of food [20, 24, 25] and have a diet higher in nutritional quality than neophobes. Thus, neophobic individuals may be more exposed to nutritional risks or suffer from specific risks related to an unvaried diet. As many studies have highlighted, sociodemographic variables are key determinants of food choices and perceptions . Food preferences and acceptance seem to be associated with food exposure . Education, occupation, and income influence the chances of exposure because they can favor a habit of dining out and knowledge of different cultures and gastronomic traditions. The association between these variables and food neophobia has been observed in several studies [31, 32] . The influence of age and place of residence has also been observed. Therefore, individuals who are more exposed to different cultures should be less neophobic. The relationship between food neophobia and general neophobia was assessed by Pliner and Hobden . Openness to different cultures seems to influence the acceptance of and willingness to try new foods. Studying this attitude in relation to food neophobia could contribute interesting information in the development of a general approach that could influence the propensity to try foods from other countries. Several survey items have been proposed by Verbeke and Poquiviqui López to study the attitudes of Belgians towards Latin American ethnic foods in an approach based on the dimensions of ethnic identity defined by Laroche et al. . Few studies have focused on these issues in reference to the Italian context. A number of studies have focused on Italians' attitudes towards Eastern European foods , on the relation between neophobia and odor identification and food preferences , and on the role of neophobia in determining food preferences . A measure of neophobia designed for Italian primary school children has also been validated . Focusing on aspects that have been shown to influence food choices is particularly interesting in a context like the Italian one, which is characterized by a deep-rooted food culture and at the same time by increasingly multicultural conditions and consequently by food polytheism.
Aims In light of the above specificities of the Italian context and of the impacts of neophobia on food preferences, this study has two objectives:
To analyze the relationships between ethnic food consumption, the psychological trait of food neophobia, and openness to different cultures in Italy; and To identify the sociodemographic characteristics of food neophobic consumers and of consumers who are open to different cultures.
Materials and Methods
Data Collection Data were collected through a national survey conducted via the computer-assisted web interviewing method (CAWI) between 8 September and 25 September 2014 by the Demetra opinioni.net firm. The sample was selected using a method that considered the stratification of the Italian population by gender and geographical area as of 31 December 2013 as determined by the Italian National Institute of Statistics (ISTAT). A sample of 1317 Italian consumers responded to a questionnaire sent to 2871 consumers for a response rate of 45.8%. The complete questionnaire includes 45 questions. A set of questions was designed to investigate ethnic food consumption habits and risk perceptions. An overview of the consumption of ethnic food in Italy and an analysis of perceptions of risk associated with this type of food are provided in a previous article . Ethnic food/products were defined as "food coming from a foreign country with gastronomic and cultural traditions different from Italian ones" . In this article, several aspects are reported to frame ethnic food consumption in Italy: Whether respondents have ever eaten ethnic food in Italy (response options: Yes and no) and the ways in which respondents come into contact with ethnic cuisine (response options: Through relatives and friends, through travel, by myself, through the mass media, and other). Food neophobia was measured with 10 items , and openness to different cultures was measured with 7 items , with response options expressed on a 1-10 Likert scale of "strongly disagree" to "strongly agree" (see Table 1 and Table 3 for response options). The original FNS was administered with a 7-point response scale. In this study, a 10-point scale was used to ensure consistency between the two measurements and the other rating scales of the questionnaire (different scales were also used by Demattè et al. and Meiselman et al. ). Ritchey et al. demonstrated through his study that excluding 2 or 4 items from the FNS improves the instrument when applied in several countries. However, we decided to maintain the original 10 items since we applied the FNS to the Italian context alone. Personal information investigated through the questionnaire included the following: Gender, age, educational qualifications, the presence of children, occupation, location, size of the city of residence, and how well the interviewee's income meets his or her financial responsibilities (see Table 2 and Table 4 for response options).
Data Analysis Descriptive statistics were applied to all variables (the sociodemographic information listed above and whether ethnic food was consumed or not) to develop a general understanding of ethnic food consumption in the Italian context. The two scales were studied over two steps. Cronbach's alpha coefficient was used to assess the internal consistency of the scales while a principal component analysis (PCA) (the maximum likelihood method with varimax rotation) was used to investigate the factor structure. The skewness and kurtosis of the two scales' distributions were also analyzed. Two indices were constructed to measure the levels of food neophobia and openness to different cultures. The two indices were calculated by summing scores allocated to the 10 statements from the FNS (range 10-100) and to the 7 statements from the openness to different cultures scale (ODCS) (range 7-70). To calculate the FNS index, the positive items were reversed . An analysis of variance (ANOVA) facilitated the study of differences in the levels of food neophobia and in the openness to different cultures on the basis of social information. Based on the FNS index, the respondents were divided into three groups: Neophilic (low scores), neutral (medium scores), and neophobic (high scores). One standard deviation from the mean was used as the cut-off point. This classification has been used in previous studies and is considered an effective method [14, 15, 32, 50] . The subjects were also divided using the same method in relation to the ODCS index. Three groups were identified: Those less (low scores), moderately (medium scores), and highly (high scores) open to different cultures. The relation between the two indices was studied by means of the Pearson correlation index. Different levels of food neophobia in relation to attitudes towards different cultures were analyzed by cross tabulation. A Chi-square test was used to verify the dependence relationship between the two variables identifying the FNS and ODCS groups. Data were processed using the SPSS (Statistical Package for Social Science) software (version 20.0) for Windows (SPSS Inc. Chicago, Illinois). The level of statistical significance was set at 5% (α = 0.05).
Results
Characteristics of the Sample The sample was 52.2% female and 47.8% male (in the Italian population, 51.5% were female and 48.5% male). Regarding regions of residence, the majority of respondents (35.2%) ). Of the respondents, 84.7% declared that they had eaten ethnic food in Italy. The majority of those who had consumed this food in Italy had come into contact with ethnic cuisine through relatives and friends (50.4%) while nearly one in four had been exposed to ethnic cuisine through their travels (24.5%), approximately one in five had been exposed to this cuisine by themselves (22.3%), and others had been exposed through the mass media (2.3%) or through other means (0.5%).
Food Neophobia The internal consistency of the FNS was assessed from Cronbach's alpha (α = 0.789). The factor structure was evaluated by means of a PCA. The scale items loaded primarily on two components, explaining 38% and 23.3% of the variance (Table 1 ). The first component loaded the regular items and is related to the levels of interest in trying new and ethnic foods while the second component loaded the reversed items and seems to be related to concerns regarding trying new foods. The mean FNS score of the Italian sample analyzed here was recorded as 51.1 (SD = 14.7, range 10-100). The FNS distribution had a skewness value of 0.08 and a kurtosis value of 0.31. The investigation of levels of food neophobia observed in relation to the consumption of ethnic food and sociodemographic variables and the results of the analysis of variance conducted are presented in Table 2 . The F-test results are statistically significant with regard to the following variables: Gender (p = 0.040), age (p = 0.000), educational qualifications (p = 0.000), the presence of children (p = 0.006), occupation (p = 0.000), meeting financial responsibilities (p = 0.000), and ethnic food consumption (p = 0.000). Levels of food neophobia were found to be higher in those who do not eat ethnic food, in males, in those older than 55 years of age, in those with no more than an elementary/lower secondary school education, in those who have children, in retirees, and in those who are experiencing many challenges in meeting their financial needs.
Openness to Different Cultures The internal consistency of the ODCS was assessed by Cronbach's alpha (α = 0.883). A PCA was used to evaluate the factor structure. The first and second principal components were found to explain 60% and 16% of the variance, respectively (Table 3 ). The first factor pertains to levels of comfort with social interactions with non-Italians while the second factor refers to language and mass media use. The mean ODCS value was recorded as 39.6 (SD = 13.9, range 7-70). The ODCS distribution generated a skewness value of 0.00 and a kurtosis value of -0.33. a Loadings that are higher on either factor are shown in bold. The results of the analysis of variance conducted are reported in Table 4 . The F-test produced statistically significant results for the following variables: Age (p = 0.000), city size (p = 0.002), location (p = 0.000), educational qualifications (p = 0.000), the presence of children (p = 0.034), occupation (p = 0.000), and ethnic food consumption (p = 0.000). More openness to different cultures was observed in those who eat ethnic food, in young adults (18-34 years of age), in those living in larger cities, in those living southern Italy or on the islands, in those with a postgraduate education, in those who are childless, and in those who are employed.
Correlations between the FNS and ODCS Indices The Pearson correlation coefficient calculated between the FNS and ODCS indices was measured as 0.312 (p = 0.000), indicating a positive but weak correlation between the two variables. For the FNS index, the subjects were divided into three groups using one standard deviation from the mean as a cut-off point: Neophilic (17%, score 10-36.4), neutral (69.2%, score 36.5-65.8), and neophobic (13.8%, score 65.9-100). In the same way, three groups were defined using an ODCS index, representing low (15.5%, score 7-25.7), moderate (67.3%, score 25.8-53.5), and high (17.2%, score 53.6-70) levels of openness to different cultures. The bivariate analysis performed on the two variables reveals that among food neophobic subjects, more than four times the number of individuals exhibit less openness than individuals exhibiting more openness. Additionally, only one of seven of those who are neophilic show lower levels of openness rather than higher levels of openness (Figure 1 ).
Discussion The ethnic food market is an important business in Italy. The consumption of ethnic products is widespread , and this trend is growing [9,10]. However, ethnic food is far from being part of everyday Italian life. The FNS and ODCS indices tested with the Italian sample present a high internal reliability coefficient. Our PCA shows that the two scales are not unidimensional and principally load on two factors. The components identified by the FNS separate the regular and reversed items, reflecting the different dimensions measured. These results are consistent with those of previous studies [41, 50] . The two components identified by the ODCS recognize the dimensions measured, social interactions, as well as language and mass media use, echoing the findings of Verbeke and Poquiviqui Lopez . The Italian sample seems to be slightly neophobic considering that the mean FNS index value is slightly higher than the midpoint of the scale. When we compare this result to the most recent FNS analysis of the Italian context, the samples analyzed by Monteleone and Proserpio seem to be less neophobic than ours and find a midpoint lower than the mean value. Although scales adopting different formats are difficult to compare, several studies suggest that a scale with more response options might produce slightly lower scores . In our study, the 1-10 scale used produced higher scores, which may be attributable to the different sampling and data collection methods applied in the analyzed studies. The data presented in this work were collected from online questionnaires of panels of Italian consumers while in the two studies listed above, participants were recruited on a voluntary basis and completed the questionnaire during experimental sessions. At the international level, samples showing lower levels of neophobia have been found in other studies [14, 16, 32, 33] while a mean value higher than the midpoint of the scale used has been reported for Lebanese students . A comparison of FNS mean scores of various countries could prove useful in providing an overview of food neophobia levels worldwide. However, generalizations and statistical comparisons are difficult to apply because the scale is sensitive to contexts, as highlighted by previous studies . The mean value of the ODCS index is instead higher than the midpoint value of the scale, suggesting a quite open sample. In analyzing the sociodemographic characteristics, we found that the description of the neophobic consumer appears to differ substantially from that of the consumer with an attitude of openness. The definition of the characteristics of consumers in relation to unfamiliar foods and different cultures deserves special attention for two main reasons. First, it fosters a comprehension of a growing phenomenon. That is, the increasing prevalence of ethnic food
Discussion The ethnic food market is an important business in Italy. The consumption of ethnic products is widespread , and this trend is growing [9,10]. However, ethnic food is far from being part of everyday Italian life. The FNS and ODCS indices tested with the Italian sample present a high internal reliability coefficient. Our PCA shows that the two scales are not unidimensional and principally load on two factors. The components identified by the FNS separate the regular and reversed items, reflecting the different dimensions measured. These results are consistent with those of previous studies [41, 50] . The two components identified by the ODCS recognize the dimensions measured, social interactions, as well as language and mass media use, echoing the findings of Verbeke and Poquiviqui Lopez . The Italian sample seems to be slightly neophobic considering that the mean FNS index value is slightly higher than the midpoint of the scale. When we compare this result to the most recent FNS analysis of the Italian context, the samples analyzed by Monteleone and Proserpio seem to be less neophobic than ours and find a midpoint lower than the mean value. Although scales adopting different formats are difficult to compare, several studies suggest that a scale with more response options might produce slightly lower scores . In our study, the 1-10 scale used produced higher scores, which may be attributable to the different sampling and data collection methods applied in the analyzed studies. The data presented in this work were collected from online questionnaires of panels of Italian consumers while in the two studies listed above, participants were recruited on a voluntary basis and completed the questionnaire during experimental sessions. At the international level, samples showing lower levels of neophobia have been found in other studies [14, 16, 32, 33] while a mean value higher than the midpoint of the scale used has been reported for Lebanese students . A comparison of FNS mean scores of various countries could prove useful in providing an overview of food neophobia levels worldwide. However, generalizations and statistical comparisons are difficult to apply because the scale is sensitive to contexts, as highlighted by previous studies . The mean value of the ODCS index is instead higher than the midpoint value of the scale, suggesting a quite open sample. In analyzing the sociodemographic characteristics, we found that the description of the neophobic consumer appears to differ substantially from that of the consumer with an attitude of openness. The definition of the characteristics of consumers in relation to unfamiliar foods and different cultures deserves special attention for two main reasons. First, it fosters a comprehension of a growing phenomenon. That is, the increasing prevalence of ethnic food not only reflects the proliferation of social change but also has important implications for the food market. Second, the group of neophobic consumers exhibiting less openness to different cultures might have a less varied diet (e.g., Arvola et al. and Schickenberg et al. ). The tendency to avoid foods that are different from those usually consumed reduces one's willingness to try healthy alternatives to known foods . This tendency also reduces one's willingness to eat certain basic foods, such as vegetable salads and fish . A diet that includes as many different foods as possible is essential to health because it ensures nutritional quality and prevents chemical accumulation due to constant exposure to the same substances . The neophobic trait is recognized as a barrier to diet modification and has a negative influence on diet quality while the willingness to accept different food alternatives can be an indicator of the ability to make positive changes in eating habits. The results of this study show that men are more neophobic than women. These findings are consistent with other studies [13, 32, 52] . However, the effect of gender on neophobia remains less clear and varies widely across studies on different regions . According to the data presented here, gender does not influence openness to different cultures. Instead, age seems to play an important role. The results of this study show that levels of food neophobia increase with age, which has already been proven by other studies [13, 52] . Openness to different cultures instead declines with age and is characterized as an attitude held by younger age groups. Other studies have highlighted that these results may seem counterintuitive given that with age, an individual should experience more exposure to a broader variety of foods and therefore develop more knowledge of different foods. These factors can promote the acceptance and willingness to try different alternatives. However, such acceptance and willingness could be attributed to a more exploratory and playful attitude even towards food, which can characterize young people, and which can promote curiosity and contact with new foods. Several studies of the Italian context have emphasized that it is precisely young people who adhere to food styles that differ from those of the Mediterranean diet and who are more open to the consumption of ethnic food . These attitudes of young people are probably favored by socialization due to the incremental likelihood of one coming into contact with different cultures relative to the past. In the Italian context, which is characterized by a culinary tradition in which food changes occur slowly, another highly interesting aspect concerns the importance of relationships to the acceptance of a new food. The majority of those reporting consuming ethnic food in Italy reported that they had come into contact with ethnic cuisine through relatives and friends. Italy has a strong gastronomic tradition, and the idea of food is linked to the idea of social relations and sharing, not only among Italians. A study recently conducted in Italy has highlighted how food appears as an element of contact and sharing between natives and migrants. Approximately two out of five immigrants interviewed stated that they have cooked their traditional dishes and have taught their recipes to Italian friends and acquaintances . The social and convivial importance of food and eating practices, which traditionally characterize Italy , is recognizable also in Italian consumers' relationship with ethnic food. Furthermore, the presence of people of different origins and traditions than the Italian ones becomes an opportunity for culinary knowledge and experimentation. Education, occupation, and income are considered variables that define the socioeconomic status of a person and that may affect opportunities to be exposed to different foods and cultures . The impact of these variables on neophobia has revealed by other studies, whose results are confirmed by the findings of this study: Consumers who are more neophobic are less educated [31, 32, 52] , of lower socioeconomic status [31, 52] , and, according to the data presented here, are retired. Conversely, consumers exhibiting an attitude of openness towards different cultures are highly educated and employed. Financial status does not emerge as a significant differentiating variable. In addition, the place of residence (urban vs. rural) was tested as a variable that could affect exposure to new products and to a multicultural environment. This hypothesis has been confirmed by other studies [27, 31, 32] . However, in this study, being an inhabitant of a large or small city or of a specific area of Italy was not found to affect food neophobia. This result could be related to unique features of the Italian Peninsula that, given its geographic position, is characterized by migratory flows throughout. Otherwise, an openness to different cultures seems to characterize residents of southern Italy and of the islands and of medium-sized cities (100,000-250,000 residents). The relationship between food neophobia, openness to different cultures, and urban living remains an aspect that deserves further study. The variable "having children" has a significant impact on both food neophobia and openness to different cultures. The most food neophobic individuals declared that they have children while those with more open attitudes were childless. This variable was analyzed because being a parent and the presence of children in the family could result in a more protective attitude and more mistrust of novelty [29, 57] . Recent studies have found that the frequency with which ethnic food is consumed is significantly correlated with levels of food neophobia [39, 50] and with degrees of openness to other cultures . Our analysis of this aspect in the Italian context presents similar results. Those reporting never having consumed ethnic food in Italy presented a higher FNS index and a lower ODCS index than those declaring that they had consumed such foods. The results of this study confirm the relationship between food neophobia and openness to different cultures as highlighted by other research [14, 39] . This study emphasizes a relationship between the psychological trait of neophobia and attitudes towards different cultures, confirming food's symbolic and cultural value. The studied aspects are immediately evident for ethnic food. These measures have become a crucial indicator of peoples' levels of willingness to accept elements of novelty in an increasingly globalized and multicultural society. This study analyzed these aspects across a nationwide sample of consumers in Italy. On the one hand, this paper extends the existing literature by providing additional empirical evidence for the Italian context, which has rarely been studied from this point of view. On the other hand, we present an apparently counterintuitive result demonstrating a tendency for young people to be less neophobic and more open to different cultures. This tendency must be studied in more depth in consideration of further social and societal variables. In fact, even if the rooted culinary Italian tradition may seem to be a deterrent to openness to new foods, the very importance attributed to food can serve as an element that encourages sociality, which seemed to emerge from this work. The acceptance or rejection of a new food involves a complex psychological mechanism that is influenced not only by personal traits but also by specific behaviors and cultural aspects. While the importance of focusing on personality traits and on food neophobia in particular has been demonstrated in several papers, a comprehensive study of these aspects is required to develop a broader account of the phenomenon in Italy as well as to compare it across different contexts. Contact with different cultures through other people, the mass media, travel, and language studies can affect openness to and acceptance of novelty and diversity . The fact that these aspects were not measured in our survey may represent a limitation of the study. Future research should therefore investigate in more depth the actual impact of these aspects in modifying consumers' attitudes. Another limitation of the study is due to the representativeness of the sample. As described in the method section, only gender and geographical area were used in the quota sampling. However the age of the sample was also consistent with that observed in the Italian population over 18 years old. Nevertheless, other sociodemographic variables (i.e., the size of the city of residence, the educational qualification, etc.) that would have made it possible to have a sample more closely related to the Italian population were not considered in the quota sampling,
Conclusions This study investigated the relation between food neophobia and openness to different cultures and the consumption of ethnic foods together with sociodemographic variables. The main results show that participants with higher levels of food neophobia do not consume ethnic food while those exhibiting higher levels of openness to different cultures consume it. Moreover, neophobic participants tend to be less open to different cultures. Some sociodemographic variables associated with food neophobia (i.e., gender, age, educational qualifications, the presence of children, occupation, and how well the interviewee's income meets his or her financial needs) and with an openness to different cultures (i.e., age, location, size of the city of residence, the presence of children, and educational qualifications) were identified. The results of this study may prove useful for those concerned with nutrition as a means to protect public health and who are tasked with designing interventions in nutritional education and communication strategies that address the traits of consumer groups who are reluctant to adopt new proposals. Considering the relation between familiarity with food and a food's place of origin for its acceptance as well as the importance of cultural and social identification , communication activities can be crucial in fostering an awareness of different foods and in promoting a varied and diversified diet. Figure 1 . 1 Figure 1. Bivariate analysis of attitudes towards different cultures and of food neophobic groups.
Figure 1 . 1 Figure 1. Bivariate analysis of attitudes towards different cultures and of food neophobic groups.
Table 1 . 1 Mean values and standard deviations of FNS (Food Neophobia Scale) items and varimax rotated factor loadings. Item Mean SD Factor 1 b Factor 2 b I am constantly sampling new and different food (R) a 5.4 2.6 0.828 -0.046 I don't trust new foods 6.9 2.3 -0.211 0.770 If I don't know what is in a food, I won't try it 5.3 2.8 0.032 0.723 I like foods from different countries (R) 4.9 2.3 0.878 -0.028 Ethnic food looks too weird to eat 6.5 2.4 -0.098 0.752 At dinner parties, I will try a new food (R) 4.4 2.7 0.858 -0.063 I am afraid to eat things I have never had before 6.5 2.5 -0.213 0.794 I am very particular about the foods I will eat 4.3 2.3 0.151 0.472 I will eat almost anything (R) 5.8 2.8 0.703 -0.037 I like to try new ethnic restaurants (R) 5.1 2.7 0.873 -0.086 % variance explained 38 23.3 a R = Reversed items; b Loadings that are higher on either factor are shown in bold.
Table 2 . 2 Food neophobia scores by sociodemographic variables. Variables N Mean (± SD) Range F p-Value Gender Male 629 51.98 (± 14.37) 14-100 4.246 0.040 Female 688 50.31 (± 15.06) 10-100 Age 18-34 35-54 297 487 48.51 (± 15.27) 50.50 (± 14.94) 10-98 14-100 10.070 0.000 >55 533 53.11 (± 14.02) 14-98 Location Northwestern Italy 346 50.07 (± 15.03) 10-100 Northeastern Italy 252 52.15 (± 15.71) 10-100 1.098 0.349 Central Italy 256 51.62 (± 16.38) 14-98 Southern Italy and islands a 463 51.02 (± 12.94) 14-94 Size of city of residence <30,000 512 52.36 (± 15.24) 10-100 30,001-100,000 365 50.46 (± 13.91) 15-98 2.548 0.054 100,001-250,000 160 51.32 (± 14.35) 10-93 >250,000 280 49.54 (± 15.01) 14-100 Educational qualifications Elementary/lower 130 54.14 (± 14.36) 14-98 secondary school diploma Vocational qualification 70 52.93 (± 15.12) 22-92 Upper secondary school 607 51.94 (± 14.29) 14-100 4.679 0.000 diploma Higher education diploma 52 53.48 (± 12.99) 24-85 Degree 387 48.61 (± 15.16) 10-100 Postgraduate qualification 72 48.52 (± 15.94) 20-100 Children Yes 804 51.99 (± 14.38) 14-100 7.451 0.006 No 513 49.72 (± 15.23) 10-100 Occupation Student/looking for first job 155 48.99 (± 14.48) 14-98 Homemaker Retired 154 244 53.20 (± 13.48) 55.37 (± 13.47) 18-91 14-98 11.129 0.000 Unemployed 138 53.36 (± 13.89) 19-94 Employed 626 48.95 (± 15.31) 10-100 Meeting financial responsibilities Very easy Quite easy 121 498 46.94 (± 16.06) 50.96 (± 14.33) 10-100 14-100 8.336 0.000 With some difficulty 545 50.87 (± 14.91) 14-100 With much difficulty 153 55.70 (± 13.64) 14-100 Ethnic food consumption Yes 1116 49.15 (± 14.26) 10-100 141.289 0.000 No 201 61.92 (± 12.63) 19-100 a Islands include Sicily and Sardinia.
lived in southern Italy or in Sicily or Sardinia followed by those living in northwestern Italy (26.3%), those living in central Italy (19.4%), and those living in northeastern Italy (19.1%) (in the Italian population, 34.5% lived in southern Italy or in Sicily or Sardinia, 26.5% in northwestern Italy, 19.8% in central Italy, and 19.2% in northeastern Italy). In total, 22.5% of the respondents were between 18 and 34 years of age, 37% were between 35 and 54 years of age, and 40.5% were more than 55 years of age (in the Italian population over 18 years old, people in the three age classes were respectively 22.3%, 37.2%, and 40.8%
Table 3 . 3 Mean values and standard deviations of ODCS (Openess to Different Cultures Scale) items and varimax rotated factor loadings. Item Mean SD Factor 1 a Factor 2 a 1. I am very comfortable dealing with non-Italians 6.6 2.2 0.879 0.184 2. I like to go to places where I can be among non-Italians 6.1 2.4 0.881 0.261 3. I like to participate in activities of non-Italians 5.9 2.3 0.883 0.295 4. Some of my friends are non-Italians 6.3 2.7 0.632 0.353 5. I often read foreign newspapers or magazines 4.4 2.8 0.293 0.850 6. I often watch foreign television 4.4 2.9 0.223 0.890 7. I like to study foreign languages 5.9 2.7 0.258 0.749 % variance explained 60 16
Table 4 . 4 Attitudes towards different cultures based on sociodemographic variables. Variables N Mean (± SD) Range F p-Value Gender Male 629 38.80 (± 13.70) 7-70 3.644 0.056 Female 688 40.26 (± 13.99) 7-70 Age 18-34 35-54 297 487 41.83 (± 13.86) 40.75 (± 13.92) 7-70 7-70 13.610 0.000 >55 533 37.22 (± 13.49) 7-70
Table 4 . 4 Cont. Variables N Mean (± SD) Range F p-Value Location Northwestern Italy 346 38.65 (± 13.30) 7-70 Northeastern Italy 252 37.72 (± 13.72) 7-70 7.649 0.000 Central Italy 256 38.23 (± 14.47) 7-70 Southern Italy and islands a 463 42.00 (± 13.72) 7-70 Size of city of residence <30,000 512 37.80 (± 13.30) 7-70 30,001-100,000 365 40.26 (± 13.31) 7-70 4.851 0.002 100,001-250,000 160 41.37 (± 16.24) 7-70 >250,000 280 40.87 (± 13.84) 7-70 Educational qualifications Elementary/lower secondary school diploma 130 35.48 (± 13.98) 7-66 Vocational qualification 70 35.47 (± 14.56) 7-70 12.253 0.000 Upper secondary school diploma 607 38.23 (± 13.18) 7-70 Higher education diploma 52 38.32 (± 13.17) 10-70 Degree 387 42.78 (± 13.56) 7-70 Postgraduate qualification 72 45.81 (± 15.41) 7-70 Children Yes 804 38.92 (± 14.09) 7-70 4.498 0.034 No 513 40.58 (± 13.45) 7-70 Occupation Student/looking for first job 155 40.50 (± 12.47) 7-70 Homemaker Retired 154 244 37.05 (± 13.78) 35.78 (± 13.12) 7-70 7-66 8.840 0.000 Unemployed 138 39.87 (± 13.57) 9-70 Employed 626 41.36 (± 14.22) 7-70 Meeting financial responsibilities Very easy Quite easy 121 498 40.71 (± 13.92) 39.79 (± 13.18) 7-70 7-70 2.480 0.060 With some difficulty 545 39.89 (± 14.31) 7-70 With much difficulty 153 36.78 (± 14.21) 7-70 Ethnic food consumption Yes 1116 40.89 (± 13.59) 7-70 70.514 0.000 No 201 32.21 (± 13.03) 7-70 a Islands include Sicily and Sardinia.
|
10.21203/rs.3.rs-1370307/v1
|
cc-by
| null | null |
openalex
|
Background: Throughout the Pandemic caused by COVID-19, health professionals have faced the ght against this disease in the front line, even without effective treatment, this uncertainty led to the presentation of various ethical crises in health services to be analyzed. Methods: An exploratory, cross-sectional investigation was developed, for which a self-application instrument was designed, in the online interview modality, aimed at doctors and university professors involved with bioethics with the purpose of analyzing ethical aspects in care of COVID-19 patients. Results: 100 interviews were collected for the convenience of their work location, 67% worked in hospitals, 11% in clinics, and 22% in universities teaching the bioethics course. Problematic situations related to high exposure to risks of health professionals, infrastructure gap, ICU beds, and hospital beds were identi ed as problematic situations. The main con icts and ethical dilemmas were related to decisionmaking for the allocation of resources, lack of culture of self-care for the health of the population, the risk of contagion from the doctor, therapies not supported by clinical trials, and patient-physician prioritization. Conclusions: It was evidenced that university doctors and teachers identi ed various problematic situations, con icts and ethical dilemmas in the care of COVID-19 patients, which compromise ethical values of maximization of bene ts, equity, in addition to ethical principles such as bene cence, justice, reciprocity, solidarity, integrity, respect, vulnerability, dignity, damaging their human and legal rights.Background In December 2019, in Wuhan China, the rst cases of easily transmitted atypical pneumonia were presented, which, shortly after, was attributed to the SARS-CoV-2 virus . The World Health Organization, faced with the rapid advance of the new disease at a global level, declared the COVID-19 Pandemic in March 2020 and since then, health services have faced this unprecedented health crisis, with a high demand for patients in critical condition, in the midst of large gaps and de ciencies in the pre-existing health systems of the pandemic, to date, more than eighty-ve million infected and more than one million eight hundred deaths have been reported worldwide. In this health emergency, the health services have shown their strengths and weaknesses, in the face of the massive need for beds, medicines, personal protective equipment and, above all, the lack of specialized professionals for the care of seriously ill patients , generating various ethical tensions, due to the urgent need to promote equality in patient care and equity in the distribution of resources in health services without any distinction. However, in this situation, health resources became insu cient even in countries with solid health systems, with the consequent collapse of public hospitals and private clinics, causing demands for maximum performance of the health professional in emergency conditions, with scarce resources and risk of contagion and death of the health personnel themselves . The World Health Organization (WHO), aware of the need to achieve equity in patient care, urged governments to make decisions to implement measures that ensure the reduction of the transmission of COVID-19 in the population, but also measures and policies to alleviate the impacts at the health systems, social and economic level , it is in this new scenario, which has revealed, the great vulnerabilities of the population and of the institutions. In the management of this pandemic health crisis, ethical guidelines and recommendations stand out under which previous pandemics have been managed, especially for triage of critically ill patients, assignment of mechanical ventilators and protection of the most vulnerable. It is worth analyzing whether these recommendations from past pandemics are su cient to face the new ethical dilemmas of this pandemic, unprecedented due to the rapid expansion at the global level, the high rates of contagion and mortality, with no cure to date, generalized inability of health care systems, health care for the excessive demand of patients reinfection and second waves of massive infections . It should be remembered that an ethical dilemma in health occurs "when a situation in the medical practice involves an operational con ict between two (or more) ethical imperatives, where applying one may mean transgressing the other, we are facing an ethical dilemma" . Among the basic ethical recommendations for the management of pandemics we nd: "to maximize the bene ts produced by scarce resources, treat people equally, promote and reward instrumental value and give priority to the most disadvantaged" . Likewise, various ethical recommendations have been given for the admission and discharge of patients in the Intensive Care Unit (ICU) in exceptional circumstances, when the health services have limited resources, among other important documents in force in any pandemic, however, we need to know if the ethical dilemmas that have arisen in all these months of the pandemic could be analyzed or adapted from these recommendations. Assistance in a pandemic context implies facing a greater and urgent demand, which always exceeds the possibilities, both in infrastructure and in materials and trained human resources, these situations would cause various con icts between health personnel and patients, who must be attended with diligence and under proper distributive justice. For all these reasons, the study was proposed, which aimed to explore the con icts and ethical dilemmas identi ed in the time of care of COVID 19 patients and propose an ethical analysis based on the ndings.
Methods
Participants and Design It is a descriptive exploratory study, which was carried out in the period between August and September, 2020 in Peru. The selection of the sample was intentional, consecutive and consisted of 100 people, who consented to participate anonymously, con dentially and voluntarily. Participants received a direct invitation via online. The questionnaire was developed for this study and the length of time that remained open the form of the online interview, closed when managed to make the sample size. The sample took into account criteria such as: geographical distribution, gender, age, scienti c discipline, eld of work, speci c area of professional work, areas of dominance and institutional a liation, among others. The basic criterion has been that of signi cance, taking into account, in addition, a criterion of heterogeneity with respect to the choice of the people summoned to offer their opinion. Doctors who were directly treating patients with COVID 19 infection in Trauma shock, hospitalization, Peruvian Intensive Care Units (ICUs), and local and international university professors of bioethics were invited. It was decided to include these two groups, considering that the eld information of doctors who work on the front line and face con icts and dilemmas is important, but also that of university bioethics teachers, who have the task of training the professional and developing competencies that allow them to identify con icts and ethical dilemmas in professional practice, incorporating ethics, "philosophy and Social Sciences in deliberation and decision-making processes" .
Variables The variables studied were: Age (years), gender (male, female, other), workplace (ICU, emergency, hospitalization of medicine, triage, university professors of bioethics), contact with patients and place of contact with COVID 19 patients, situations that arise in the health system and con icts and ethical dilemmas that occur in times of pandemic.
Statistical analysis For data analysis, the statistical program IBM SPSS version 26 was used. To describe the quantitative variables, the distribution of frequencies and averages were used.
Ethical analysis In the ndings, the ethical problems and con icts that were raised by the interviewees were identi ed and later the moral values that come into tension and that form the ethical dilemma were contrasted by spinning the contextual arguments and ethical re ection based on ethical principles and discussion of the results with experts in bioethics in the region.
LIMITATIONS The data was collected by Google Forms and responses were only accepted until reaching 100 questionnaires, so we could have some selection bias, however, the results could be useful for new studies.
Results The sample consisted of 100 participants, in terms of gender, 59% were women and 41% men, the average age of the respondents was 40.19 ± 11.1 years. Regarding the workplace, 67% worked in hospitals, 11% in clinics and 22% in universities teaching the bioethics course (see table 1 ). The situations that occur most frequently in the health system in the COVID 19 pandemic are high exposure to risks from health professionals, lack of human resources, lack of personal protective equipment, exposure to high viral load, discrimination and mistreatment of doctors, little national research on COVID19, infrastructure gap, lack of ICU beds and hospital beds (see table 2 ). The interviewees identi ed as very frequent or frequent the ethical dilemmas related to con icts in the triage of patients and the availability of beds, in saving the greatest number of lives and collapsed health services, the con ict between the effort of doctors to care for patients and the lack of a culture of selfcare for the health of the population, the con ict of values between the duties of the doctor to save life and the risk of life of the doctor himself due to the lack of adequate personal protective equipment and environmental work conditions, con icts between the specialty of the doctor and the functions he exercises in the context of a pandemic in patient care (see Table 3 ).
Discussion Ethical issues are inexorably present in all phases of a pandemic and in situations of disasters and catastrophes, hence the importance of developing studies on this unprecedented health emergency due to its magnitude and rapid global expansion. The present study was developed to identify the perception of the main ethical dilemmas and situations that arise in the care of COVID 19 patients. Among the most frequent problematic situations in health services, those that directly affected health professionals were identi ed as high exposure to occupational risks, lack of personal protective equipment (PPE), exposure to high viral load and lack of wage payments. Occupational exposure added to lack of services could be a fatal combination for health professionals who serve on the front line, since it has been demonstrated a greater risk of transmission in personnel during their exposure to aerosols of patients hospitalized for COVID 19 , hence the importance of minimizing contagions and providing su cient PPE and of adequate quality to ensure less exposure (21) , since poor quality PPE could give false security to health personnel with the consequent contagion and death . The results of the study showed that the doctors had had, throughout this health emergency, episodes of discrimination and abuse. These results are similar to those found in various studies on attacks on health personnel before the pandemic and under the context of the pandemic , as well as discrimination and stigmatization, which in some cases materializes in workplace violence due to the panic of the general population at contagion by health personnel , this situation being a violation of human rights under the defense of human rights, with serious repercussions on the mental and emotional health of the victims . It has been observed that health personnel in critical and non-critical areas suffer from burnout , stress , anxiety and depression . The results also showed problematic situations related to the de ciencies of the health system such as health infrastructure gaps, lack of specialized human resources to care for patients in critical condition, little attention to public health, among others, this situation re ects severe problems in the social determinants of health and health inequities, especially in rural regions, poverty areas, jungle regions and provinces far from large cities, where levels of inequity and social injustice are evident, as pointed out by Withers, Mellissa . Governments in pandemic made decisions such as social isolation, protection of vulnerable persons and widespread use of masks, to mitigate the spread while preparing health services for care hospital mass of patients many critics, however on this path full of urgent needs, they neglected primary health care, with public health in charge of ensuring the common good, eliminating inequities, preventing neglect of other diseases, and promoting social justice. In our results, we also evidenced a perception of little research in Peru on COVID-19, in the face of a context of great and multiple uncertainties that require urgent responses through research, such as the lack of effective treatment and a vaccine, ignorance the extent and duration of natural immunity after a SARS-CoV-2 infection, as well as the reasons for higher mortality in ethnic minorities , lack of effectiveness in containment campaigns and self-care of health, need for access to molecular tests and medical devices, as well as other problems that have increased the vulnerability of people, such as the greater frequency of mental health problems, family violence, change in educational systems, loss of employment, poverty and many others that may require evidence-based responses through collaborative national research. However, it is imperative that, in the context of the pandemic, quality research is generated that guides decision-making based on scienti c evidence and with the use of data under a framework of human rights and ethical principles ; Therefore, it is necessary for the Research Ethics Committees, despite the accelerated review times, to be attentive to any attempt to exploit vulnerable groups or exploitation of their data (Sorokin et al. 2020 ), exposure to inappropriate risks , breach of the con dentiality of patient data, they must analyze the ethical justi cation, scienti c validity and social value of each research protocol on COVID-19, no relaxation of ethical or scienti c criteria can be admitted and even more if it is about trials with vaccines against this fearsome disease . On the other hand, our results suggest that several of the problematic situations are related to public health, the management and allocation of resources, which should be analyzed with values such as solidarity, reciprocity, well-being, social justice and equity; using these approaches, we can achieve achievements for society as a whole, without individualistic positions , as established in the Universal Declaration of Bioethics and Human Rights of UNESCO (2005): "the fundamental equality of all people must be respected human beings in dignity and rights, in such a way that they are treated with justice and equity " . In the study, various con icts and ethical dilemmas were identi ed under the context of COVID 19, such as those related to the efforts and great di culties to maximize the bene ts of the care of these patients, compared to health services with scarce resources; These situations forced immediate decision-making that involved ethical principles related to: seeking the greatest good for the greatest number of patients (utilitarianism), the allocation of ventilators based on the patient's need (equality, solidarity, justice ), protection and respect for the patient's will to accept or reject invasive treatment (autonomy, freedom) and in some cases decisions were made to prioritize patients according to social bene t. The interviewees identi ed as frequent con icts and dilemmas: con ict between the effort of doctors to care for patients and the lack of culture of self-care of the population's health, con ict of values between the doctor's duties to save life and the risk of life the doctor himself due to lack of adequate personal protective equipment and work environment conditions, con icts between the doctor's specialty and the functions he performs, dilemmas about prioritizing the allocation of scarce resources, con ict between the treatment plan and the information without scienti c support or therapies not yet proven with clinical trials mainly. Next, some of these con icts and ethical dilemmas are analyzed based on ethical values of pro t maximization, equal treatment, as well as ethical principles such as bene cence, justice, reciprocity and solidarity. 1. Decision-making to prioritize patients in their care and life support measures in the ICU: The Pan American Health Organization (PAHO) has also added ethical guidelines for this pandemic for "the use of limited resources in critical health services , recommending prioritization with transparent criteria to generate trust in the population and decrease burdens on health professionals, it also recommends saving the greatest number of lives, prioritizing patients with better clinical conditions to survive treatment in addition to palliative care for those who do not access treatment, as well as treating people fairly . Robert, René 2020, in an article referring to ethical dilemmas in intensive care units, recognizes that various ethical questions arise when intensive care physicians must make painful decisions contrary to their basic ethical principles in the face of the high demand of patients in services without immediate availability of beds . Decisions in the context of the pandemic have been di cult and unavoidable, both in the prioritization of patients to be hospitalized in ICU beds, as well as in the withdrawal of life support treatments; these situations have mainly confronted doctors on the front line, to face serious ethical problems; that have forced the choice of different alternatives to guarantee a greater community good, such as the prioritization of patients with the greatest probability of surviving COVID 19, selection of those who have lived fewer stages of life, who have a social utility, fewer comorbidities , among others, many of these criteria have been observed, especially age, so Ruth Macklin rejects "age or age ranges" as a decisive factor for the allocation of ventilators in the event of a criterion tie in the context of pandemics . Robert et to the., In the article "Ethical Dilemmas due to the pandemic of the Covid 19" identi es ten elements to support the decision of the intensivists to admit or not a patient in the ICU, based on scores of fragilities, comorbidities, knowledge the patient's advance will, evaluation of the patient's previous or estimated quality of life, as well as the importance of collegiate decision-making and consultation with experts . It should be noted that, in most Latin American countries, advance directives are not regulated despite the pandemic, which in this context could protect and respect the autonomy of patients affected by severe COVID-19 and avoid procedures invasive not approved by the patient. Some recommendations, guidelines and provisions have been given to support decision-making with utilitarian approaches in the allocation of ICU hospital beds in a state of catastrophe comparable to the current pandemic, however, some of these use prognostic criteria in addition to in the long term and functional status, the exclusion of groups of patients with certain serious comorbidities, this restriction would compromise the principle of justice, it is therefore necessary that all patients have the chance of being eligible. 2. The con ict between the efforts of physicians to care for patients and the lack of a culture of self-care for the health of the population: It should be noted that, in the region, an attitude of self-medication and of pre-hospital medication has been added in the general population, which in many cases has devastating effects such as delay in hospital care, serious adverse reactions, worsening disease, among others . The health professional has a low incidence in the face of the desperate actions of the population, whether it is motivated by the lack of care given the saturation of the services, the perception of the population of dying in hospital services and the lack of specialized professionals for care, the event with the greatest weight in the population has been the scene of corpses in the streets or on the outskirts of hospitals, followed by queues of patients trying to be admitted to hospital services. The health professional is limited to exercise his role as treating physician, in the face of the overwhelmed measures of the population seeking immediate attention. The various information media have an impact on the population's self-medication, which is why reports from professionals have tried to spread the word about the risks of using drugs with serious side effects. Professional ethics only affects this level as a guild that supports the ethical guidelines of the medical prescription and the precautionary form with which the instructions for the use of the medication must be given. Given the controversies that occurred in the region, there were governments that supported giving medication from pharmaceutical products whose scienti c evidence does not exist for the treatment of patients with COVID-19. 3. The con ict of values between the duties of the doctor to save life and the risk of life of the doctor himself: In this pandemic, an exacerbation of occupational risks has been observed in health personnel, with the constant threat of being infected during the execution of invasive procedures that generate aerosols to save the lives of their patients with COVID 19, it adds to this, working in environments with exposure to patients with high viral load and with di cult access to personal protective equipment . Under this context, it is that many doctors around the world, complying with the Hippocratic Oath and the principle of bene cence, have been infected or have died victims of SARS-CoV-2 and its complications . Many doctors, in addition, continue to work in COVID services, although they do not have related specialties, but due to the lack of human resources, they had to leave their usual services, to be able to work in COVID units. 4. The con ict between the treatment plan and the unscienti c information circulating on the networks and the con ict between the efforts of doctors to treat patients: In the context of this health emergency, the general population, health systems, governments and academics have faced uncertainty, which has brought many urgent questions, few answers and even information without scienti c support, which circulates and frightens the general population. Decisionmaking based on evidence requires valid information, the product of fast and e cient processes, that comply with a neat development and deep respect for ethical principles and recommendations, scienti c validity , compliance with national and international regulatory frameworks, scienti c integrity. It is also required to encourage and nance research with social value and that the information generated is at the service of the population. Non-male cence as part of medical practice has been forgotten as a principle that should guide the technical competence of the health professional, in the absence of scienti c evidence where the practice in each context of treatment of COVID-19 patients builds daily experience for therapeutic management. 5. Decision making to prioritize the patient health personnel in their care and life support measures in ICU). In this regard, PAHO also "Prioritize those who have the task of saving the lives of others: People such as health providers risk their lives to save the lives of others. Prioritizing their care therefore responds to a criterion of justice, and also allows saving the greatest number of lives given their central role in caring for others" (PAHO 2020). The prioritization of doctors for their care in critical units also implies reciprocity towards health professionals in which society rewards the professional to put themselves at risk to help patients without any discrimination and in many cases working with demand and demand that exceed their possibilities. The health workers must receive support for priority access to COVID-19 vaccines too , therefore, the prioritization of health personnel is related to the instrumental value of their profession, since they are the ones who are struggling in the different services health against this fearsome disease and it is most likely that after recovering their health, they will continue in that same role. In this regard, several questions arise, such as the perception of the discrimination community of the social value of each patient, but also doubts within the health system itself, such as: what type of doctors to prioritize? Who prioritizes? and under what criteria? In this decision-making, there could be inequity scenarios, for example: Do we owe greater reciprocity to the doctors who care for COVID patients on the rst line, such as the areas of triage, ICU, trauma shock and COVID hospitalization? would they be left out or should prioritization be extended to physicians who support diagnosis and treatment, directly as the area of tomography, dialysis, etc.? or Should all general practitioners in the community contagion phase be included, where every patient with any pathology could also be infected with COVID? Faced with these questions, the proposal of Bianchini Alahi Dana; Rivera López, Eduardo Enrique; Luna, Florence; Alegre, Marcelo; Perez, Diana Inés and Ignacio Mastroleo, to share decisions: "this kind of decision is not only the responsibility of doctors or hospital authorities, but, ultimately, the community in general and each person interested in the particular case" likewise, they conclude on the need to take the support of an approved legal framework, of speci c norms on the subject . Ezequiel Emanuel and team, in the article called: "Fair Allocation of Scarce Medical Resources in the Time of Covid-19" adapt ethical recommendations to COVID 19: On "the maximization of bene ts" as a utilitarian value, which implies "saving the greatest number of individual lives or save patients with more years of life after treatment", Ezequiel proposes a random selection of COVID 19 patients or in order of arrival, to avoid unequal treatment between patients . Due to the rapid progress of the pandemic, hospitals globally were already preparing to face di cult situations such as the imbalance between the demand for clinical care and the effective availability of ICU beds, hospitalization, among others . In the present study, the interviewees also identi ed the dilemma related to (decision making to prioritize the patient health personnel in their care and life support measures in ICU), regarding this situation, Persad G, Wertheimer A and Emanuel E. in an article published in The Lancet called "Principles for allocation of scarce medical interventions" , they justi ed the prioritization of health personnel in a pandemic situation by assigning them an "instrumental value" by recognizing the moral importance of "those who can saving others, or rewarded those who have saved others in the past . Ezequiel Emanuel emphasizes the need for a "multi-value ethical framework that can be adapted, depending on the resource and the context" .
Conclusions And Recommendations The doctors and university teachers of the analyzed sample identi ed various situations of frequent ethical problems due to the high exposure to risks of health professionals, lack of human resources, lack of personal protective equipment, and exposure to high viral load and how ethical dilemmas frequent con icts in the triage of patients and the availability of beds, and saving the greatest number of lives in the face of a situation of few resources in the care of COVID-19 patients. Ethical dilemmas were analyzed from the values of pro t maximization, equity, as well as ethical principles such as bene cence, social justice, distributive justice and solidarity, especially in marginalized communities. Insu cient infrastructure conditions, low equipment capacity in ICUs, and non-specialized professionals to deal with the management of COVID-19 patients are a characteristic of health systems pre-existing to the pandemic, as well as conditions of limited access to health in marginalized communities, the risk being that these situations of injustice persist or worsen after the pandemic due to the worsening of the social and nancial situation . In this scenario, university doctors and teachers are facing adversities under which they must make dilemmatic decisions contrasting moral values to protect health against a pandemic that is undermining the economic and social balance of the region. The ethical recommendation guides have the possibility of building a framework for re ection to guide the decision-making that the treating physician must inevitably make in the face of COVID-19 patients and future pandemics. The study was approved by the Institutional Research Ethics Committee of the Universidad Católica de Santa María, reference number CIEI (193-2020), it also complies with the ethical principles of the Declaration of Helsinki, the sensitive data of the participants has been protected.
Consent for publication This study has consent for the publication of results. Abbrevations CIEI Institutional Research Ethics Committee COVID- 19 : 19 Abbrevations CIEI Institutional Research Ethics Committee
|
10.1001/jama.1907.02530190052015
|
public-domain
| null | null |
openalex
|
given below.1 They show that Phenol Sodique contains some¬ thing like 0.5 or 0.06 per cent, of phenols, dissolved in about 0.75 per cent, of sodium hydroxid. In other words, it appears to be essentially a very dilute alkaline solution of some im¬ pure coal-tar product, presumably of crude carbolic acid. The analysis could not profitably lie carried further, because the amount of the antiseptic agent is so very small.2 The consideration of this analysis, in connection with the claims made for Phenol Sodique, leaves little doubt as to one reason for the secrecy concerning its composition; although no educated physician could be deceived into believing for a moment that Phenol Sodique could fulfill the promises of its promoters, even if it were "the best antiseptic, hemostatie and disinfectant on the market," as the manufacturers say in their advertisements. From its composition, it can only have the very moderate and ordinary antiseptic qualities of a dilute phenol or cresol solution, modified only to a very slight extent by the free alkali. According to the manufacturers, however, "Phenol Sodique is a wonderful preparation." Just how wonderful appears from these extracts from the dissertations in the pamphlet which is enclosed in the package. Note particularly the matter which we have put in capitals : "Catarrh. Old Colds, etc.: Drink every morning and even¬ ing a glass of water containing ten to thirty drops of Phenol Sodique. . . "SMALLPOX: TO PRETEXT ATTACK take internally three or four times a day. fifteen or twenty drops of Phenol Sodique in one tablespoonful of sugar and water. "Measles, Scarlatina and Erysipelas: Same treatment as for Smallpox. "TYPHOID FEVER: TO PREVENT ATTACK take internally three or four times a day, fifteen or twenty drops of Phenol Sodique. "CHOLERA : TO PRETEXT spread sawdust or sand, wet with Phenol Sodique. in apartments. "THE VERY BEST PRECAUTION is to drink, morning and evening, a glass of water containing from fifteen to thirty drops of Phenol Sodique. This is the kind of therapeutics and prophylaxis taught to flic medical profession by their self-appointed instructors, the proprietors ! But this matter has a serious, as well as a ludicrous, side: What is the proper epithet to apply to those who. knowingly and intentionally, impress on the ignorant lay public that one can with impunity expose himself to smallpox, cholera, typhoid or scarlet fever, or measles, by taking a few drops of very dilute carbolic acid, or by sprinkling a little on sawdust ? What must be the consequences to those who trust in these assurances? And what should be the lawful penalty for those whose blunted moral instincts permit them wilfully to endan¬ ger the lives of others for a little financial gain? It would be interesting to know the real opinion of the responsible members of the firm of Hance Bros. & White on these ques¬ tions. The Montyon Prize was awarded by the French Institute in 1861-forty-six years ago-how many victims a year? 1. Examination of Phenol Sooiqce.-Phenol Sodique is a black, strongly alkaline liquid, having a cresol-like odor. No carbon dioxid is set free on addition of acids. On evaporation and igni¬ tion a residue remains behind which contains sodium. The product gives the characteristic tests of phenols ; because of the small amounts present the distinction of cresols from phenol was not attempted. On evaporation, a tarry residue remains. The follow¬ ing determinations were made : SPECIMEN A. Lab. analysis. Specific gravity (at 15.5C.).1.014 1.011 1.012 Phenols (bromin absorption calculated to phenol) .05 .44 .59 Residue (evaporation residue dried 10 hours at 100C .).2.56 2.02 2.10 Sodium hydroxid (evaporation residue charred, leached, and its alkalinity cal¬ culated to NaOH) .70 .81 .84 2. Phenol Sodique (II. B. & W.) must not be confused with similarly named products of the pharmacopeias and formularies, since these contain a very much greater percentage of phenol. For instance, the "Phenol Sodique" of the National Formulary (Liquor Sodii-Carbolatis) contains 50 per cent, of pure phenol. I. e.. it has about a hundred times the strength of the proprietary preparation. Drug Journals and Dr. McCormack's Address. San Francisco, Cal., Oct. 25, 1907. To the Editor:\p=m-\Inotice that many of the drug journals are attacking rather strongly Dr. McCormack's remarks before the American Pharmaceutical Association, or rather that portion of them in which he stated that the National Association of Retail Druggists had representatives at some of the state legislatures, opposing desirable legislation. N. A. R. D. Notes, for October 17, has a long article on this subject, which is mainly made up of a general denial of the charges, and an attack on Dr. McCormack. Of course, I can not speak for any other state, but so far as California is con- cerned, Dr. McCormack is perfectly correct, and the truth of his attitude has been pointed out twice in the California State Journal of Medicine; once in April, 1905, and again in October, 1907. During the session of the legislature of 1905, the repre- sentative of N. A. R. D. Notes, Mr. Cheatham, had prepared and introduced a bill amending the law regulating the practice of medicine, which, had it passed (at no time was there any danger of its passing), would have completely destroyed the value of our state medical law. Not only was this action accepted by the N. A. R. D. as an act of its official representa¬ tive, but the publication of this association-Y. A. R. D. Notes, printed, I believe in March, 1905, or thereabouts, a very self- laudatory article, in which, referring to this bill of Mr. Cheat- ham, it said: "We commend this bill to the consideration of the druggists of every state that have legislative fights on their hands, and counsel them that at times it is wise and necessary to fight fire with fire." The last expression is used because the avowed purpose of the bill introduced by Mr. Cheatham was to distract the attention of physicians from certain pure food and drug bills which had been introduced in the legislature. Incidentally, I may advise you that Mr. Cheatham's pernicious activity-whether it originated with him or was inspired by the home office of the N. A. R, D., I do not know-was entirely unnecessary, for the reason that the drug bills in question were not properly drawn, were not sup¬ ported bv any considerable number of our profession, and would not have passed. Philip Mills Jones. Sectarianism and the Philadelphia County Medical Society. Pittsburg, Pa., Oct. 26, 1907. To the Editor:\p=m-\InThe Journal, October 26, page 1448, I notice with pleasure that you record that the Philadelphia County Medical Society has introduced a resolution to admit all reputable practitioners of whatever school. In Pittsburg we have been doing this for several years. In 1905 our county society (Allegheny County) adopted ordinances and by-laws providing that: "Any legal regular practitioner of good moral character and professional standing, who is willing to subscribe to the Code of Ethics of the American Medical Association, shall be eligible as a candidate for membership in this society, without any restriction as to time or college of graduation, or time of residence in the county, other than ample time to allow the censors to investigate his character and standing." The first graduate of a homeopathic school was admitted into membership in 1905 and since then several others have come in. We would have it distinctly understood, however, that homeopaths, etc., are not eligible, but that we welcome physi¬ cians irrespective of school or college, so long as they fulfill the requirements of Section 3 of our ordinances as quoted above, and cast off forever any "trade-mark" which they may have had. We also feel that this same spirit should permeate the en¬ tire county and should be adopted by every county medical society. Edward B. Heckel. Philadelphia, Oct. 28, 1907. To the Editor:-May I be permitted to correct the errone¬ ous report of your Philadelphia correspondent (The Journal, October 26, page 1448) concerning the recent action of the Philadelphia County Medical Society in amending the section of its by-laws relating to qualifications for membership. On the blank form of application is printed the amendment, as Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 diluted in an ounce of water. ..."
|
10.1007/s11139-016-9836-7
|
cc-by
| null | null |
openalex
|
Given a Weil-Deligne representation of the Weil group of an l-adic number field with coefficients in a domain O, we show that its pure specializations have the same conductor. More generally, we prove that the conductors of a collection of pure representations are equal if they lift to Weil-Deligne representations over domains containing O and the traces of these lifts are parametrized by a pseudorepresentation over O.Introduction The aim of this article is to study the variation of automorphic and Galois conductors in p-adic families of automorphic Galois representations, for instance, in Hida families and eigenvarieties. We relate the variation of Galois conductors in families to purity of p-adic automorphic Galois representations at the finite places not dividing p and for the variation of automorphic conductors, we use local-global compatibility. We establish the constancy of tame conductors at arithmetic points lying along irreducible components of p-adic families. 1.1. Motivation. In [Hid86a, Hid86b] , Hida showed that the p-ordinary eigen cusp forms, i.e., the normalized eigen cusp forms whose p-th Fourier coefficients are p-adic units (with respect to fixed embeddings of Q in C and Q p ), can be put in p-adic families. More precisely, he showed that for each positive integer N and an odd prime p with p ∤ N and Np >= 4, there is a subset of the set of Q p -specializations of the universal p-ordinary Hecke algebra h ord (N; Z p ), called the set of arithmetic specializations, such that there is a one-to-one correspondence between the arithmetic specializations of h ord (N; Z p ) and the p-ordinary p-stabilized normalized eigen cusp forms of tame level a divisor of N. It turns out that the tame conductors of the Galois representations attached to the arithmetic specializations remain constant along irreducible components of h ord (N; Z p ) (see [Hid88, Theorem 2.3, 2.4] ). Following Hida's construction of families of ordinary cusp forms, further examples of families of automorphic Galois representations are constructed, for instance, Hida families of ordinary automorphic representations of definite unitary groups, families of overconvergent forms (see the works of Hida [Hid95] , Coleman, Mazur [CM98] , Chenevier [Che04] , Bellaïche, Chenevier [BC04] et. al.). The aim of this article is to understand the variation of automorphic and Galois conductors in these families of automorphic Galois representations. Since the restrictions of p-adic automorphic Galois representations to decomposition groups at places outside p are known to be pure in many cases, we focus on the variation of conductors in families of pure representations of local Galois groups. 1.2. Results obtained. Let p, l be two distinct primes and K be a finite extension of Q l . Let O be an integral domain containing Q. In theorem 3.1, we show that given any Weil-Deligne representation of the Weil group W K of K with coefficients in O, its conductor coincides with the conductors of its pure specializations over Q p . Next, in theorem 3.2, we show that a collection of pure representations of W K over Q p have the same conductor if they lift to Weil-Deligne representations of W K over domains containing O and the traces of these lifts are parametrized by a pseudorepresentation T : W K → O. The eigenvarieties are an important source of examples of families of Galois representations. The traces of the Galois representations attached to the arithmetic points of an eigenvariety are interpolated by a pseudorepresentation defined over the global sections of the eigenvariety. Unfortunately, this pseudorepresentation does not lift to a Galois representation over the global sections. However, by [BC09, Lemma 7.8.11], around each nonempty admissible open affinoid subset U, the pseudorepresentation defined over the global sections of an eigenvariety lifts to a Galois representation on a finite type module over some integral extension of the normalization of O(U). But this module is not known to be free over its coefficient ring. So theorem 3.2 cannot be applied to eigenvarieties to study the tame conductors of all arithmetic points. To circumvent this problem, we establish theorem 3.4 which can be used to study the tame conductors of a large class of arithmetic points. Theorem 3.1, 3.2, 3.4 apply to p-adic families of automorphic representations, for instance, to ordinary families, overconvergent families, and explain the variation of the tame conductors. We illustrate it using the example of Hida family of ordinary automorphic representations for definite unitary groups (see theorem 4.1).
Preliminaries For every field F , we fix an algebraic closure F of it and denote by G F the Galois group Gal(F /F ). For a finite place v of a number field E, the decomposition group Gal( E v /E v ) is denoted by G v . Let W v ⊂ G v (resp. I v ⊂ G v ) denote the Weil group (resp. inertia group) and Fr v ∈ G v /I v denote the geometric Frobenius element. The fraction field of a domain A is denoted by Q(A) and the field Q(A) is denoted by Q(A). If p is a prime ideal of a ring A, then the mod p reduction map is denoted by π p . The integral closure of a domain R in Q(R) is denoted by R intal . For each map f : R → S between domains, we fix an extension f intal : R intal → S intal of f . Let q denote the cardinality of the residue field k of the ring of integers O K of K. Let I K denote the inertia subgroup of G K . Let {G s K } s>=-1 denote the upper numbering filtration on G K by ramification subgroups. Fix an element φ ∈ G K which lifts the geometric Frobenius Fr k ∈ G k . The Weil group W K is defined as the subgroup of G K consisting of elements which map to an integral power of the geometric Frobenius element Fr k in G k . Its topology is determined by decreeing that I K is open and has its subspace topology induced from G K . Define v K : W K → Z by σ| K ur = Fr v K (σ) k for all σ ∈ W K . Definition 2.1 ([Del73, 8.4.1]). Let A be a commutative domain of characteristic zero. A Weil-Deligne representation of W K on a free A-module M of finite rank is a triple (r, M, N) consisting of a representation r : W K → Aut A (M) with open kernel and a nilpotent endo- morphism N ∈ End A (M) such that r(σ)Nr(σ) -1 = q -v K (σ) N for all σ ∈ W K . The operator N is called the monodromy of (r, M, N). Suppose (r, V, N) is a Weil-Deligne representation with coefficients in a field L of characteristic zero which contains the characteristic roots of all elements of r(W K ). Let r(φ) = r(φ) ss u = ur(φ) ss be the Jordan decomposition of r(φ) as the product of a diagonalizable matrix r(φ) ss and a unipotent matrix u. Following [Del73, 8.5], define r(σ) = r(σ)u -v K (σ) for all σ ∈ W K . Then (r, V, N) is a Weil-Deligne representation (by [Del73, 8.5]) and is called the Frobenius-semisimplification of (r, V, N) (cf. [Del73, 8.6] ). It is denoted by V Fr-ss . Definition 2.2. If (r, V, N) is Weil-Deligne representation of W K on a vector space V over an algebraically closed field of characteristic zero, then its conductor is defined as cond(r, V, N) = V I K ,N =0 + ∞ 0 codimV G u K du where V I K ,N =0 denotes the subspace of V on which I K acts trivially and N is zero. Lemma 2.3. Let f : A → B be a map between domains of characteristic zero. Let E (resp. F ) be a field containing A (resp. B). Let ρ : G → GL n (A) be a representation of a group G. Then the E-dimension of the space of G-invariants of the representation G ρ -→ GL n (A) ֒→ GL n (E) and the F -dimension of the space of G-invariants of the representation G ρ -→ GL n (A) f -→ GL n (B) ֒→ GL n (F ) are equal if G is finite. Proof. If r : G → GL n (F ) is a representation where F is a field of characteristic zero, then the dimension of the space of G-invariants of r is equal to the trace of the idempotent operator 1 |G| g∈G r(g). Since f (n) is equal to n for any integer n, the lemma follows.
Main results
Denote W K → GL n (O) be a Weil-Deligne representation of W K with coefficients in O. If f : O → Q p is a map such that f (r, N) is pure, then the conductor of f (r, N) is equal to the conductor of (r, N) ⊗ O Q(O). Proof. By [Sah14, Theorem 4.1], there exist positive integers m, t 1 , , t m , unramified char- acters χ 1 , , χ m : W K → (O intal ) × , representations ρ 1 , , ρ m of W K over Q cl with finite image such that ((r, N) ⊗ O Q(O)) Fr-ss ≃ n i=1 Sp t i (χ i ⊗ ρ i ) /L , (f (r, N)) Fr-ss ≃ n i=1 Sp t i (f intal (χ i ⊗ ρ i )) /Q p So the dimension of ((r, N) ⊗ O Q(O)) I K ,N =0 (resp. (f (r, N)) I K ,N =0 ) over L (resp. Q p ) is equal to n i=1 ρ I K i (resp. n i=1 (f intal ρ i ) I K ). By lemma 2.3, the L -dimension of ((r, N) ⊗ O Q(O)) I K ,N =0 is equal to the Q p -dimension of (f (r, N)) I K ,N =0 . Note that G u K is contained in I K for any u >= 0 and r(H) is finite for any subgroup H of I K . Hence dim L ((r, N)⊗ O Q(O)) H is equal to dim Q p (f (r, N)) H by lemma 2.3. This shows that the conductor of f (r, N) is equal to the conductor of (r, N) ⊗ O Q(O). Now we establish an analogue of the above result for pseudorepresentations of Weil groups. The notion of pseudorepresentations was introduced by Wiles [Wil88] in the two-dimensional case, and by Taylor [Tay91] in full generality. They are defined by abstracting the crucial properties of the trace of a group representation. Theorem 3.2. Let O be an integral domain and res : O ֒→ O be an injective map. Let T : W K → O be a pseudorepresentation of dimension n >= 1 and let (r, N) : W K → GL n (O) be a Weil-Deligne representation such that res T = trr and f (r, N) is pure for some map f : O → Q p . Then there exists a non-negative integer C such that the following statement holds. If (r ′ , N ′ ) : W K → GL n (O ′ ) is a Weil-Deligne representation over a domain O ′ satisfying (1) res ′ T = trr ′ for some injective map res ′ : O ֒→ O ′ , (2) f ′ (r ′ , N ′ ) is pure for some map f ′ : O ′ → Q p , then cond (r ′ , N ′ ) ⊗ ′ Q(O ′ ) = cond (f ′ (r ′ , N ′ )) = C. Proof. Note that by [Sah14, Theorem 5.4, Proposition 2.8], there exist positive integers m, t 1 , , t m , unramified characters χ 1 , , χ m : W K → (O intal ) × , representations ρ 1 , , ρ m of W K over Q cl with finite image such that ((r ′ , N ′ ) ⊗ O ′ Q(O ′ )) Fr-ss ≃ m i=1 Sp t i (res ′intal (χ i ⊗ ρ i )) for any Weil-Deligne representation (r ′ , N ′ ) : W K → GL n (O ′ ) over a domain O ′ satisfying condition (1), (2). Then lemma 2.3 shows that the conductors of (r ′ , N ′ ) ⊗ O ′ Q(O ′ ) and ⊕ m i=1 Sp t i (χ i ⊗ ρ i ) are equal. So by theorem 3.1, the result follows by taking C to be the conductor of ⊕ m i=1 Sp t i (χ i ⊗ ρ i ). Let F be a number field and T : G F → O be a pseudorepresentation such that T = T 1 + + T n where T 1 : G F → O, , T n : G F → O are pseudorepresentations. Using [Tay91, Theorem 1], choose semisimple representations σ 1 , , σ n of G F over L such that trσ i = T i for all 1 <= i <= n. ρ i | Ww = cond WD n i=1 σ i | Ww . Proof. By [Sah14, Theorem 5.6, Proposition 2.8], there exist positive integers m, t 1 , , t m , unramified characters χ 1 , , χ m : W K → (O intal ) × , representations θ 1 , , θ m of W K over Q cl with finite image such that WD (⊕ n i=1 ρ i | Ww ) Fr-ss is isomorphic to ⊕ m i=1 Sp t i (π intal m loc intal (χ i ⊗ θ i )) and WD (⊕ n i=1 σ i | Ww ) Fr-ss is isomorphic to ⊕ m i=1 Sp t i (χ i ⊗ θ i ). Then lemma 2.3 shows that the conductors of WD (⊕ n i=1 ρ i | Ww ) and WD (⊕ n i=1 σ i | Ww ) are equal to the conductor of ⊕ m i=1 Sp t i (χ i ⊗ θ i ). The result follows.
Conductors in families In this section, using the example of Hida family of ordinary automorphic representations for definite unitary groups, we show how results of section 3 describes the variation of tame conductors in p-adic families. Let F be a CM field, F + be its maximal totally real subfield. Let n >= 2 be an integer and assume that if n is even, then n[F + : Q] is divisible by 4. Let l > n be a rational prime and assume that every prime of F + lying above l splits in F . Let K be a finite extension of Q l in Q l which contains the image of every embedding F ֒→ Q l . Let S l denote the set of places of F + above l. Let R denote a finite set of finite places of F + disjoint from S l and consisting of places which split in F . For each place v ∈ S l ∪ R, choose once and for all a place v of F lying above v. An irreducible constituent π of the G(A ∞,R F + ) × v∈R Iw( v)-representation S λ,{χv} (Q l ) is said to be an ordinary automorphic representation for G if π U (l b,c ) ∩ S ord λ,{χv} (U(l b,c ), O K ) = 0 for some integers 0 <= b <= c (see [Ger10, Definition 2.2.4, §2.3] for details). Let U be a compact open subgroup of G(A ∞ F + ), T be a finite set of finite places of F + containing R ∪ S l and such that every place in T splits in F (see [Ger10, §2.3] ). Let T ord denote the universal ordinary Hecke algebra T T,ord {χv } (U(l ∞ ), O K ) (as in [Ger10, Definition 2.6.2]). Let Λ be the completed group algebra as in [Ger10, Definition 2.5.1]. By definition of T ord , it is equipped with a Λ-algebra structure and is finite over Λ. An O K -algebra homomorphism f : A → Q l is said to be an arithmetic specialization of a finite Λ-algebra A if ker(f | Λ ) is equal to the prime ideal ℘ λ,α (as in [Ger10, Definition 2.6.3]) of Λ for some dominant weight λ for G and a finite order character α : T n (l) → O × K . By [Ger10, Lemma 2.6.4], each arithmetic specialization η of T ord determines an ordinary automorphic representation π η for G. An arithmetic specialization η of T ord is said to be stable if WBC(π η ) is cuspidal. Let m be a non-Eisenstein maximal ideal of T ord (in the sense of [Ger10, §2.7]). Let r m denote the representation of G F + as in [Ger10, Proposition 2.7.4]. Then by restricting it to G F and then composing with the projection GL n (T ord m ) × GL 1 (T ord m ) → GL n (T ord m ), we get a continuous representation G F → GL n (T ord m ) which is denoted by r m by abuse of notation. Since m is non-Eisenstein, the G F -representations η r m and r πη are isomorphic for any arithmetic specialization η of T ord m (by [Ger10, Proposition 2.7.2, 2.7.4]). Theorem 4.1. Let w ∤ l be a finite place of F , a be a minimal prime of T ord m and m be the maximal ideal of T ord containing a. Suppose m is non-Eisenstein. Denote the quotient ring T ord m /a by R(a) and the representation r m mod a by r a . Then there exists a non-negative integer C such that the conductor of WD(r πη | Ww ) is equal to C for any stable arithmetic specialization η of R(a). Moreover, the conductor of WBC(π η ) w is also equal to C for any such specialization η. Proof. If π is an irreducible constituent of the G(A ∞,R F + )× v∈R Iw( v)-representation S λ,{χv} (Q l ) such that WBC(π) is cuspidal, then for any finite place w of F not dividing l, r π | Gw is pure by [Car12, Theorem 1.1, 1.2] and the proofs of theorem 5.8, corollary 5.9 of loc. cit. Note that r a | Ww is monodromic by Grothendieck's monodromy theorem (see [ST68, ). So theorem 3.1 gives the first part. Since local Langlands correspondence preserves conductors, the rest follows from [Car12, Theorem 1.1] on local-global compatibility of cuspidal automorphic representations for GL n . the fraction field of O by L and the algebraic closure of Q in O by Q cl . Theorem 3.1. Let (r, N) :
Fix a finite place w of F not dividing p and assume that O is a Z p -algebra.Definition 3.3. The irreducibility and purity locus of T 1 , , T n is defined to be the collection of all tuples of the form(O, m, κ, loc, ρ 1 , , ρ n ) where O is a Z p -algebraand it is a Henselian Hausdorff domain with maximal ideal m, κ denotes the residue field of O and is an algebraic extension of Q p , loc : O ֒→ O is an injective Z p -algebra homomorphism and for each 1 <= i <= n, ρ i is an irreducible G F -representation over κ such that the trace of ρ i is equal to loc T i mod m and ρ i | Gw is pure. Theorem 3.4. Suppose the irreducibility and purity locus of T 1 , , T n is nonempty and the restrictions of σ 1 , , σ n to W w are monodromic. Then for any element (O, m, κ, loc, ρ 1 , , ρ n ) in the irreducibility and purity locus of T 1 , , T n , we have cond WD n i=1
For v ∈ R, let Iw( v) be the compact open subgroup of GL n (O F v ) and χ v be the character as in [Ger10, §2.1, 2.2].Let G be the reductive algebraic group overF + as in [Ger10, §2.1]. For each dominant weight λ (as in [Ger10, Definition 2.2.3]) for G, the group G(A ∞,R F + ) × v∈R Iw( v) acts on the spaces S λ,{χv} (Q l ), S ord λ,{χv} (O K ) (as in [Ger10, Definition 2.2.4, 2.4.2]). For an irreducible constituent π of the G(A ∞,R F + ) × v∈R Iw( v)-representation S λ,{χv} (Q l ),let WBC(π) denote the weak base change of π to GL n (A F ) (which exists by [Lab11, Corollaire 5.3]) and let r π : G F → GL n (Q l ) (as in [Ger10, Proposition 2.7.2]) denote the unique (up to equivalence) continuous semisimple representation attached to WBC(π) via [CH13, Theorem 3.2.3].
|
10.7596/taksad.v6i5.1267
|
cc-by
| null | null |
openalex
|
This paper describes the aspects of Turkish postmodernism of the "second wave", which is justified by an insignificant number of theoretical works and inadequate elaboration of studies concerning the representatives of Turkish postmodernism "second wave". The exception is represented by the works of Russian turkologists M.M. Repenkova and O.V. Kareva, who consider the features of the postmodern paradigm in their studies within the novelistics of the leading representatives of the Turkish postmodernism "second wave". The representatives of the "second wave" (Hasan Ali Toptaş, İhsan Oktay Anar, Perihan Mağden, Kürşat Başar, etc.) were not the object of close research before, and therefore the consideration of their creativity as the next stage in the development of Turkish postmodernism is relevant. The "second wave" of postmodernism is characterized by the appeal to the literature of symbolic modeling in 1990s. During this period, the literature of postmodernism develops into a powerful trend, which becomes the predominant trend of the cultural life of Turkey in 2000s. It is worth noting that, if in the postmodern prose of 1980s was marked by the dominance of "lyrical stream", then in 1990s and 2000s the dominant trend was represented by narrative modification. Hasan Ali Toptaş, known for his mastery of language use, is regarded as one of the main representatives of the second wave of postmodern literature.Introduction It should be noted that the priorities of the Turkish readers and critics who were brought up within the previous sharp-social literature are not changing immediately. In 1980s the works of postmodernists of the "first wave" produce the effect of a "burst bomb", but they remain misunderstood as a whole, are rejected by the reading public and literature researchers. Only in 1990s the attitude towards postmodernism begins to change gradually. During this period, with a general negative attitude toward this "Western phenomenon, harmful to national literature" critics, for example, already have an awareness that it needs to be studied. Then, there are also the first serious literary studies on the topic of postmodernism in 1990s by F. Akatlı, G. Aytaç, J. Agevit, A. Kabaklı, B. Moran, F. Nadjy, etc. (Repenkova, 2010) . Postmodern romances-palimpsests become fashionable not only among the intellectual elite of Turkish society, but also among the urban educated population as a whole. The representatives of the "first wave" in postmodernism are replaced by prose writers of the "second wave" who turn to the literature of symbolic modeling during 1990s. (I.O. Anar, H.A. Toptaş, P. Mağden, K. Başar, N. Genç and others). The postmodern prose of that period developed into a powerful literary trend, which turned into a mainstream cultural life of the country during 2000s. At the same time, if the "lyrical stream" (B. Karasu, N. Eray, etc.) dominated in the Turkish postmodernist prose during 1980s, then the narrativistic modification prevails during 1990s -2000s (I.O. Anar, H.A. Toptaş, P. Cure, etc.) (Kareva, 2014) .
Materials and methods The materials for the study were the materials of the biography and creative activity of writers, as well as postmodern novels that enable to identify the works by Hasan Ali Toptaş and Parihan Mağden as the representatives of Turkish postmodernism of the "second wave" during the analysis. The following research methods were used in the process of the work analysis: Psychoanalytic method to understand subconscious and unconscious behavior of novel characters; Biographical method establishes the links between an author's biography and novel features; And postmodern method, in which a novel is understood as a discourse, and the artistic image is replaced by a simulacrum.
Results and discussion H.A. Toptaş (born on October 15, 1958) is an outstanding Turkish writer. His first book of short stories "The Identity of a Smile" (Bir Gülüşün Kimliği) was published in 1987. A well-known Turkish scholar Yıldız Ecevit in his work "Postmodernist expansion in Turkish literature" (Türk Romanında Postmodernist Açılımlar) calls him "modernist postmodern", as well as "Kafka in Turkish literature" (Ecevit, 2016) . Hasan Ali Toptaş, the winner of the literary award by Cevdet Kudret in 1999 for the novel "Thousand grim pleasures" (Bin Hüzünlü Haz), which is, on the one hand, a postmodern story in terms of pluralism, metaprose and intertextuality. On the other hand, it contains many Kafkaesque elements in terms of absurd, surreal and paradoxically secular reality description. Hasan Ali Toptaş, who is considered one of the leading authors of modern Turkish literature, draws attention by his style, in particular. Academic studies and criticism in the novels by Toptaş usually emphasize this unique poetic style. However, its origins and structure have not been analyzed yet. Toptaş strengthened more his poetic style in his two last novels "Thousand grim pleasures" (Bin Hüzünlü Haz) and "The East Drowsens" (Uykuların Doğusu) and thus he transformed the narrative discourse into a literary image. Despite the postmodern features, which are usually attributed to the writer, Toptaş style takes its roots in modernism in accordance with the concept of reality and a reader's assumption. The author focuses on the complex and contradictory reality of an individual and presupposes a qualified / a wellprepared reader regarding the concept of reality and the transformation of a reader's assumption in modernist aesthetics. The discourse was isolated from the interference of the conscious "I" to some extent and aimed at the manifestations of consciousness reflections in the novels by Toptaş. Narrative discourse, which is far from unity and continuity, indicates space and time with extraordinary aspects, accumulating grotesque and surreal images. Thus, an imitative unity of a word and a thing is destroyed, and the concept of reality becomes an aesthetic representation of existence in mind. Linguistic discourse strategies that transform each component of a narrative into a literary image are the main results of this study. The novel "Waste" (Heba) is the first book by H.A. Toptaş translated into English. It tells the story of a middle-aged anti-hero Turk Ziya Kul, persecuted by the tragic death of his wife and the unborn child, as well as an experienced military terrorizing the Syrian border. The story by Toptaş about Ziya Kul trauma, depicts the silent erosion of the hero's soul in a chaotic world, controlled by the mentality of the crowd, poverty, ignorance and cruel, aggressive military culture. The center of the novel is represented by Mr. Ziya's final and tragic attempt to find peace in life. But the inner turmoil, suffering and anguish of the protagonist will follow him everywhere, and his transfer to the Edenic village Yazıköy will not save him. Sleep, memory and reality flow into each other in the narrative when feelings go out of order (smells are heard, sounds are seen). This is most clearly expressed in the first third of the book, in which a reader is exposed to almost 100 pages of Ziya's dreams. A popular and versatile Turkish writer H.A. Toptaş cultivates his own style. His narrative technique, especially his use of metaphor, dreams, allegories and folklore narrative can be fascinating sometimes, but some of these technical and stylistic components remain problematic. In addition to its invigorating lifelessness ("It's so, but it's very strange," said Mr. Ziya, puzzled, "It's impossible that you know all this") (Toptaş, 2014) , he manages to treat the reader with a feeling close to contempt, providing clear words with superfluous adjectives and adverbs: "Just do not lie there, time to get up!" Mrs. Nurgül said in a reproachful voice; "He exclaimed being in a shock:" They found me!" (Toptaş, 2014) . This is exactly the type of the letter that appears when indulgence towards a reader is combined with some disbelief due to language. One page has such diverse lines as "lose your mind", "straw that broke the camel's back", "at the end of our acute mind", "do not know the boundaries", "the sands of time", "rush like the wind". They are joined by all kinds of thoughtless buildings (birds have "beady eyes", houses "buzz like a hive", people wait "with bated breath" and exchange with "knowing views") (Toptaş, 2014) . Also the book has a lot of "sudden" actions. Sometimes we are told about the surprise twice, as in the sublime sentence "suddenly they all stopped abruptly" (Toptaş, 2014) . Sometimes things get even more immediate: "His mind suddenly slipped to the Syrian border, and then, just as suddenly, a shaft of light slipped" (Toptaş, 2014) . Thus, the novels by Hasan Ali Toptaş are filled with a unique cultivated poetic style. His postmodern code, and especially the use of metaphor and allegory, is not clear to every reader. Speaking of the "Second Wave" of postmodernism in Turkish literature, one is doomed to pay attention to the work by Perihan Mağden. Orhan Pamuk, the well-known writer of Turkey who won the Nobel Prize in Literature in 2006, told about P. Mağden as "one of the most inventive and outspoken writers of our time" in the Guardian (June 3, 2006) . O. Pamuk emphasizes the author's manner and style, "the way she twists and turns the Turkish language, the delight she takes in the thrust and pull of popular culture, and her brilliant forays into subjects that everyone thinks about these have earned her the love of her readers and the respect of her fellow writers" (ibid.). Her novel "Escape" (Biz Kimden Kaçıyorduk Anne) published in 2007 was considered one of the best Turkish novels of that year. Her previous novels were the following ones: "Two girls" (İki Genç Kızın Romanı), "The murders of messenger boys" (Haberci Çocuk Cinayetleri) and "Companions" (Refakatçi). Her latest collection of non-fiction literature is called "Political Essays". The book by P. Mağden "The murders of messenger boys" is a detective of absurdity. The inhabitants of the city have bizarre names and have no less bizarre occupations. The city does not have a name; the inhabitants correlate it with a labyrinth by themselves. In this city a genetic experiment was carried out many years ago to deduce the ideal messenger boys. Due to special drugs, these boys look like six-yearolds, although in fact they can be much older. They reason like small robots. The protagonist, who returned from the journey, has to take up the investigation of these messenger boys murders. The story about the murders is only the part of the plot, which can be classified as magical realism. The novel was translated into many languages. The book by P. Mağden attracts the attention of "advanced" literature fans. The main meaning of the novel is not the investigation of local pride murders -artificially created impeccable boys-messengers. Main thing in the novel is the absurd atmosphere of the labyrinth city, where everything around is not what it seems. "Milet Books" publishing house, calling P. Mağden "a popular and an innovative Turkish author", describes the novel as "a gloomy, comic, irreverent and soporific story, the study of humanity infinite absurdity and its futile attempts to create perfection skilfully wrapped in the mystery of a murder" (Hamer, 2009) . Maureen Freely responds to the novel "The murders of messenger boys" as follows in the magazine about the Turkish culture "Cornucopia": "The actions of the novel take place in the city that feels like Russian, but inhabited by the residents with Chinese names, full of apathy of the nineteenth century, but with the references to Hollywood and overshadowed by the villainous fertility expert, it is difficult to classify, it is impossible to suppress" (Freely, 2009) . P. Mağden is the writer who deals with journalism. She writes about politics, military affairs, human rights, homosexuality and the general human condition in Turkey. She was tried and acquitted because she called for the permission to refuse from military service because of moral principles.
Conclusion The "second wave" of Turkish literary postmodernism is a new trend in the literature of Turkey at the turn of the 20th and the beginning of the 21st century. The representatives of this group are a small group of writers who started their creative activity in the 1990s of the 20th century. The "second wave" of postmodernism is characterized by the appeal to the literature of symbolic modeling in 1990s. The literature of postmodernism develops in a powerful current, which becomes the predominant trend of the cultural life of Turkey in 2000s. At the moment, Turkish literature is in a constant development and popularization, which contributes to the increase and the diversity of themes and techniques used by the authors. Having studied the stages of postmodern trend emergence and development, we can draw the following conclusions: The discoverer of the Turkish postmodernism was Oguz Atay with the novel "The Expendables" (Tutunamayanlar) in 1970. At that time, the intellectuals of the Turkish novelists did not know postmodern or modern literature. Therefore, Oguz Atay's novel provoked conflicting criticism. The next novel, which turned out to be equally incomprehensible to critics, was Orhan Pamuk's novel "The Black Book" (Kara Kitap), released in 1990. Even though O. Pamuk's novel was recognized in European countries, and Turkish critics took a different look at social realism. After that, postmodernism acquired a different perception and became a popular trend in Turkey. Thus, after the study of postmodernist writers of the "second wave", we came to the conclusion that their creative style is difficult and incomprehensible in some cases for an unprepared reader. At the moment, Turkish literature is in a constant development and popularization, which contributes to the increase and the diversity of themes and techniques used by the authors. This was facilitated by the coup d'etat in Turkey in September of 1980. The country changed its view on the structure of secular society, which was hardly perceived by Turkish writers and gave rise to a feeling of despair and pessimism in the creative environment. At the same time, during this period there was an active informatization and computerization of the Turkish people. All this contributed to the emergence and the development of Turkish postmodernism. Hassan Ali Toptaş, İhsan Oktay Anar, Perihan Mağden, Kürşat Başar, and Elif Shafak turn to such postmodern methods as pluralism, metaprose, intertextuality, multilevel nature, magical realism and tabulation in their works. By their works they raise acute social issues. The writers of the second wave resort to narrative modification and symbolic modeling.
|
10.19026/rjaset.6.3801
|
cc-by
| null | null |
openalex
|
Performance evaluation is the most central part of the enterprise performance management process. The nature of administrative staffs' work and the work features are different. The results of their work are difficult to assess. Thus, how to assess their performance becomes the top priority of the enterprise management. In the study, data evaluation index determination, calculation of the process of design and evaluation criteria are discussed. The study also constructs a SME executive's performance appraisal data model. A wide range of assessment methods are used in the design process of the model which comprehensive quantitative evaluates staffs' work performance. The assessment information of the employee's basic performance is put in EXCEL spread sheet to performance sort which is simple and convenient and examination results are fair and impartial, easy to convince the public. In the model, no absolute values are set in assessment times, assess results fully consider the teamwork effect. Moreover, it's easy for examiners to use for lateral longitudinal contrast, employees will find their own strengths and weaknesses and know the reasons timely which can improve their assessment scores as well as help their colleagues. In general, it easily gives accurate guidelines for future work.INTRODUCTION SME refers to enterprises that the number of employees is less than 1000 and more than 100. Administrate staffs is staffs engaged in enterprise services, supervision, coordination who not directly engaged in production activities but play vital roles in safeguarding the realization of the strategic objectives, the creation of corporate interests. Their work cover A wide range, including planning, procurement, human resources management, financial management, quality management and public affairs (Lu and Sun, 2007; Avila et al., 2010; Xu, 2011) . According to their job duties, Administrate staffs can be roughly divided into several types which are staffs who provide decisionmaking information to senior managers, who provide advice to the business sector and other functions of the office and coordinate between the departments. Their work has characteristics as following (Korman, 1986; Boyle, 2001) : Difficult to quantify: Administrate staffs' work is very similar that monthly operating basic procedures of operation and most of the work results is difficult to be determined by qualitative indicators, rather than quantitative data to measure and subjectivity is difficult to be avoid. The outcome is not exploitations: Most of the word content is courses of certain events and the impact of their contribution to the company is very indirect and difficult to judge the quality of their work. Work is not easy to plan: Most of the administrate staffs' day-to-day work are temporary tasks. In some enterprises it even accounts for more than 40% of the total work which directly affect the assessment focus. Difficult to our performance: Large amount of temporary teamwork exist within the administrative departments, especially when in busy time or temporary tasks are arranged. Most or all personnel in the sector are often involved in a job, administrative personnel performance's difficulties in the team work greatly increase. An increase in coordination: Administration work needs to do a lot of coordination of internal and external. Measure of the quality of their work involves not only administrative staff themselves, may involve multiple assessors. The requirements of their quality are relatively high: Due to the requirements of the job duties, administrative requirements are higher than other employees in the cultivation of knowledge, morality and the ability to work. Generally, they have higher educations, most of who are colleges or undergraduates. Performance evaluation refers to the a comprehensive, systematic, scientific considerations assessment process of staffs' behavior, performance and results through the scientific application of qualitative and quantitative methods according to the needs of corporate human resources management in a certain period of time (Jones, 2001) . Enterprise business people could examine their performance. However, some non-business sectors, such as administrative departments, whose members are located in different administrative positions which covers a wide range and are complex hierarchical. Moreover, there are many differences in nature and characteristics of their work which led to that assessing its performance have become imperative in enterprise management. Following phenomena occur in the current management of SME executives (Fernandez-Araoz, 1999; Grimsley and Jarrett, 1973) : Above problems in the performance appraisal make enterprise performance appraisal work to achieve its proper role in the enterprise staff recruitment, reward and punishment, executive training cannot achieve the desired effect. Many executives of assessment held an indifferent attitude, thus forming a vicious loop affecting the realization of corporate objectives.
MODELING Assessment contents and indexes: Virtue: This mainly refers to the loyalty of the staffs of the company which mainly evaluate the performance of the ideological and political performance and professional ethics. Based on principles of practical, easy inspection, operational, four evaluation aspects should be designedobedience, the ability to actively implement the competent superiors arrangement, service, whether service objects are satisfy with their own job, responsibility, whether on their own work and temporary assigned by task best, make themselves and their superiors satisfied from beginning to complete the job and have the courage to take responsibility for the mistakes encountered the work, team awareness whether working harmony with superiors and colleagues and have the overall positive and proper cooperation. Capability: This mainly refers to the administrate staffs' ability, technology and knowledge required by job. This focused on the evaluation of business knowledge whether the staff has a wealth of job management knowledge to understand the work processes and business, master job content and have the ability of independent processing services, can satisfactorily deal with business issues, innovation whether professional knowledge and the ability originally and independently to solve problems, the ability to execute whether successfully work toward the goal, communication skills whether actively communicate with colleagues, whether there is clear and concise, methodical skills as well as quickly and efficiently to contact with the relevant departments and personnel to solve the problem of coordinating capacity. Diligence: This mainly refers to the degree of the dedication of the administrative staff which focuses on evaluation of the administrative staffs' attendance-daily attendance and record of employees' late arrive, early leave, personal leave or sick leave, absenteeism, not lazy and not burnout, initiative-without direction or order of superior one can take the initiative to improve the working methods, work completion rate-whether one complete job content evaluated according to the percentage of completion of work, diligence and professionalism-whether one has consolidate the professional ideology and has a strong sense of professionalism, a strong awareness of enterprising, selfless dedication, diligent working attitude. Results: Mainly it refers to the efficiency and performance of the staffs. The evaluation focuses on the number of staffs-whether tasks are completed on time in accordance with company policy, the quality of work-whether in accordance with company policy tasks are completed in highquality, the effect of work that award-winning of the work and whether the work has been completed to achieve the aim set, cost control-the ability to 1 ). Following are the Fig. 1 : Assessment standards setting: Two types of standard should be considered when performance standards are set: o Basic standards: Basic standards mean the level an assessment is expected to reach and it can be achieve through the staffs' efforts. Moreover, for certain jobs, the basic standards can be listed. o Standard of excellence: The standard of excellence is the performance level that there are no requirements and expectations, but one can achieve. The excellence level is not everyone can --------------------------------------------------------------------------------------------------------
Choosing assessment methods: Qualitative indicators of the evaluation: With reference to the principles and requirements of the key event evaluation method and the 360 performance evaluation method, different evaluators from their own prospect assessed the assess object. Thus, the evaluation results reflect the behavioral characteristics and performance of the administrative staffs in different scenarios, different aspects which consolidated to have a more comprehensive and objective evaluation of the administrative staffs. Assesse are divided into four categories: leaders' evaluation, colleagues' evaluation, service objects' evaluation and selfevaluation (Peng, 2004; Qiao, 2008) . Leaders directly in charge of the administrative personnel and other heads of the related business departments can first evaluate the performance level of the administrative staff according to the administrative staff personal annual performance. Then the performance level can be converted to the corresponding fraction values from which the administrative staff annual leading evaluate performance scores are derived. Other employees of the department and other business related degree sector employees-first assessed administrative staff performance level based on the annual performance of administrative staff personal. Then the performance level can be converted into the corresponding fraction values from which the administrative staff annual colleague's evaluation performance scores are derived. First, evaluation objects complete personal summary of the work. Then, select typical representative four times the number of evaluators from various departments of the enterprise. The evaluation last convened evaluation on behalf of the forum and to consider individual summary of material approved performance level, the performance level converted into the corresponding score summary from which other personal annual job performance evaluation scores are drawn. First, assess the performance level according to own annual performance. Then the performance level can be converted into the corresponding score from which the annual work e = n i n i i Z X X X 1 Rank , , , , where, e is the sub-annual performance evaluation point, Rank is the EXCLE sort function, Xi is enterprise administrative staff's individual performance point. {Xi Xn} is a performance evaluation point collection of entire administrative staff Involved; Z is the performance sort which is descending. Specific procedures: Rank function returns specified value rank of the field of the result set partition-collection performance point of the entire administrative staff. The rank is related to the line before the (descending order value) plus one. Corresponding to descending the performance of corporate executives, performance point is given, such as six people who all have performance value participate in the performance evaluation and who score highest performance points compared with the corresponding 6, the minimum is 1 as Table 3 . Excellent performance calculation model: e e = n i n i i Z Y Y Y 1 , , , , Rank where, i Y = ) : ( bn bi i e e Average X , n Y = ) : ( bn bi n e e Average X , Average is the EXCLE arithmetic mean of function. i Y Is the excellent performance that administrative staff performance value minus the value. { i Y n Y } is an excellent performance evaluation point collection of entire administrative staff Involved; Z is the performance sort which is descending. The specific procedure is the same as basic performance value calculation process shown in Table 4 . Specific to say is: ) ( M e E i + ) (C e i + ) (D e i + ) (P e i where, 1 ) (M e i = ) (M e b + ) (M e e 。 2 ) (C e i = ) (C e b + ) (C e e 。
。 The proportion of the modeling is shown in Fig. 3 .
ALGORITHM EXPERIMENT Qualitative indicators of appraisal calculation: First, assess the performance level of the administrative staff from four perspectives: leadership, colleagues, clients and self-evaluation. Then draw all levels of administrative personnel performance scores and then get the average value each grade. Thus, administrative staff's performance points are got, respectively.
Basic performance: The leader's performance evaluates point: Put the leadership evaluation summary performance score value in descending order. Then the corresponding ranking is the performance leader evaluation points of the administrative staff in the year Colleagues performance evaluates point: Put the departmental staff performance evaluation score values in descending order, Then the corresponding ranking is the point of evaluating performance of the administrative staff of the year from colleagues Service objects performance evaluates point: Put administrative staff performance evaluate scores of the annual service objectin descending order. Then corresponding ranking is the point of evaluating performance of the administrative staff of the year from service objects Self-evaluation of performance points: Put performance evaluation of the self-evaluation score in descending order. Then corresponding ranking is the administrative staff annual self-evaluation of performance points Add the aforementioned four performance point, namely the basic performance of the executives "virtue" and "sense of responsibility" Excellent performance: Minus administrative staff's annual performance point to the department's annual average basic performance point. Remove negative numbers; get the points in descending order. Corresponding ranking is the year of the administrative staff's excellent performance in "virtue" and "sense of responsibility". Virtue" and "sense of responsibility "performance: The aforementioned basic performance and performance excellence sum is the performance of the administrative staff's "virtue" and "sense of responsibility "performance. Qualitative evaluation in performance evaluation indicators of administrative staff, such as service, obedience, team awareness virtue, business knowledge, innovation ability, the ability to execute, communication skills (capacity), initiative, diligence and professionalism (diligence) can be done in accordance with this Act.
Evaluation of quantitative indicators calculated: Attendance basic performance point: Based on the unit attendance record, aggregate as the number of attendance. Put the aggregation in descending order that the corresponding ranking is attendance basic performance point of administrative staff of the year. The attendance excellent performance point: Minus the sum of the administrative staff attendance basic performance point to the entire administrative staff year average attendance performance point. Remove negative numbers, the put the points in descending order. The corresponding ranking attendance excellent performance point of administrative staff of the year.
Attendance performance point: Add the aforementioned two-part performance points which is administrative staff attendance performance points of the year. Indicators in the performance evaluation of corporate executives, such as "indicators of the quantitative evaluation of attendance, work completion rate in diligence, work effect, the number of tasks, task quality, cost control in performance can be assessed in accordance with this Act. Balance to determine the rank of annual performance evaluation of administrative staff: Sort above four areas-virtue, capability, diligence and achievements-performance points and rank them that can be derived the administrative staff performance ranking within departments in the year. If two staff has the same performance points, results of Deneng Peregrine to it can determine the annual performance evaluation of the administrative staff ranking according to the weights order-achievements, virtue, capability and diligence. Economic globalization has brought unprecedented opportunities to our country's enterprises, but also brought huge challenges. To cope with the challenges Enterprises need to continuously upgrade its own strength. Corporate administrative staff is an integral part of the corporate management team groups which is an auxiliary and coordination role for the production, management. Its quality and effectiveness is the key of enterprise management level and efficiency. The core purpose of the performance appraisal is a comprehensive study of the ability and level of staff. Effective performance appraisal system can improve the performance of their work, develop the potential of employees, employee career development and enhance their competitive advantage, which to some extent determines the competitiveness of enterprises. The development and implementation of the performance appraisal system for corporate executives related to the future destiny of the enterprise. Enterprises must integrate theory with practice and constantly improve the practice of administrative personnel evaluation mechanism so as to improve the overall level of work, establish positive values, creating more profits for the enterprise, improve operational efficiency and build competitive advantage. Therefore, the establishment and improvement of scientific performance evaluation mechanism specific for administrative staff can help comprehensive, accurate and objective evaluate their work performance, which stimulate enterprise administrative staff's enthusiasm, improve service quality and has an active role in promoting the improvement of enterprise management level. This will help administrative staff complete administrative objectives and work plans in highquality, achieve the common development of enterprises and individuals.
CONCLUSION According to the characteristics of the nature of the administrative staffs' work, the study has designed a data model for performance evaluation: n i n i Z X X X e 1 1 , , , , Rank and be calculated by basic performance (eb) and excellent performance (ee). Then get (eb+ee) that can be the result of administrative staff's annul performance evaluation. comprehensively quantitative evaluate personnel work performance by using the combination of the application of the diversification of assessment methods, such as 360-degree assessment method (assessment of virtue), method of management by objects (assessment of capacity), behavioral observation scale method (assessment of diligence), key performance indicators act (assessment of results). In the appraisal process, appraisal staff only required to fill in the personal performance information which can also be collected directly by the assessment office before assessment. The data will by input to EXCEL spreadsheet by the assessment staff. This can come up with the sort of performance results which to ensure the result is fair and impartial. The results also give full consideration to the effect of teamwork. Through the evaluation, employees understand that in order to get higher scores help improve colleagues is also an important factor. Of course, due to the limited capacity, the complete system performance modeling work has not been completed. Till now, Simply a EXCEL RANK function helps. Goal setting qualitative assessment of the corporate executives and the use of scientific assessment methods still have shortcomings. Overall, performance accounting is slightly cumbersome that it should be further streamlined and refined. Also, diligence assessment still could be improved. Fig. 1 : 1 Fig. 1: Analysis diagram of the administrative staffs' performance evaluation model in SME control operating cost in reasonable range plan (Table1). Following are the Fig.1:
Fig. 2 : 2 Fig. 2: Content and weight qualitative indicators of performance evaluation of the administrative staff department performance evaluation of the self-evaluation value is derived. All this can be shown in Fig. 2 Quantitative indicators of appraisal: Classify and aggregate corresponding administrative staffs' quantitative indicators values o The establishment of the evaluation model:According to the principles of benchmarking requirements, we can build a performance appraisal data model as follows combined with the characteristics of enterprise management:
Fig. 3 : 3 Fig. 3: The SME administrative staff performance evaluation data model proportion 3 ) (D e i = ) (D e b + ) (D e e 。
Evaluation positioning is not clear, no specific content managers stipulated time table circles and hooks and add some reviews. Then he/she will give a brief communication with each subordinate on the contents of the table and sign in each examination sheet at last that the evaluation work is done. The evaluation results in addition to the individual special circumstances, if any, production, safety, quality of major accidents was named as incompetent, other personnel monotony are be classify competent, which has greatly restricted the effect of evaluation work. No feedback of the evaluation results, the lack of a performance improvement plan. Leaders of most SMEs don't make related evaluation and analysis of the evaluation results and don't propose appropriate improvements, which makes the correct behavior of assessed persons cannot get the organization upper affirmation, the inadequacies competent leaders can't be pointed out and corrected and also do not know how to improve in order to meet the expectations of the organization. The indicators are difficult to quantify, performance evaluations are unfair. Assessment of corporate executives contains a large number of qualitative assessment indicators that lack objectivity. So called emotional points come out that the evaluations are susceptible to the influence of personal prejudices and make unrealistic evaluation that it would not be true pictures of its features and the true level. This can damage enthusiasm of the real hard working, capable administrative staffs and will affect the fairness of the performance appraisal. Performance appraisal has emphasized too much on the history and less on development potential. Enterprise performance appraisal is at the level of "backward-looking and ignores the forward- looking development, does not pay attention to scientific predictions of human potential and the development trend analysis, lack the function of analyzing and proposing recommendations for improvement of the short comes of the administrators' work. Therefore, it is difficult to determine the focus of future training and direction of development. Also, it is difficult to tap its potential and improve their ability. Performance appraisal merely is a formality and stimulating is difficult. Enterprise performance appraisal process is generally the same: first, fixed forms are distributed to various departments, : In the past, most of the performance evaluations of corporate executives are based on their performance over the past year. But clear quantitative criteria are lack that it's difficult to carry out the actual evaluation of results of the work and behaviors, performance and quality characteristics affect the results of the work. That's to say, either subjectively evaluation of the evaluation staffs or appraisal staffs are involved in a negative attitude that they are irresponsible to make evaluations.
Table 1 : 1 Administrative personnel performance evaluation index in the Table1 First level indexes Second level indexes Code Meaning D1 Whether one takes his own work and dedicates to temporary tasks assigned to him, satisfies his superiors, from start to finish to complete the job, has the courage to take responsibility of the mistakes encountered the work Service sense D2 Assessment the satisfaction of the service object of their own work Obedience D3 Ability to actively implement the arrangements of the competent superiors Team spirit D4 Be harmony with superiors and colleagues, work based on the overall situation, to cooperate actively and properly Capability Business knowledge N1 Whether the staff has a wealth of job management knowledge to understand the work processes and business, master job content and have the ability of independent processing services, can satisfactorily deal with business issues Innovation capability N2 Whether professional knowledge and the ability originally and independently to solve problems. Ability to execute N3 Whether successfully work toward the goal Communication skills N4 Whether actively communicate with colleagues, whether there is clear and concise, methodical skills as well as quickly and efficiently to contact with the relevant departments and personnel to solve the problem of coordinating capacity. Diligence Attendance Q1 Daily attendance and record of employees' late arrive, early leave, personal leave or sick leave, absenteeism, not lazy and not burnout. Q4 Whether one has consolidate the professional ideology, and has a strong sense of professionalism, a strong awareness of enterprising, selfless dedication, diligent working attitude. Performance Work results J1 Whether tasks are completed on time in accordance with company policy. Number of tasks J2 The number of tasks completed by the staff Quality of tasks J3 Whether high quality completion of tasks in accordance with company policy Operating cost control J4 Control operating cost in reasonable range plan.
Table 2 : 2 Award-winning corresponding performance point list Corresponding performance points Award-winning level (or ranking)
Table 3 : 3 Descending the performance of corporate executives Performance Basic Evaluation appraisal Efficiency performance subjects value Ranking point Worker 1 60 4 4 Worker 2 80 5 5 Worker 3 50 3 3 Worker 4 10 1 1 Worker 5 30 2 2
Table 4 : 4 Excellent performance calculation model Performanc Excellent Excellent Assessme e appraisal performan Performan performance nt object value ce value ce ranking rate Worker 1 60 14 2 2 Worker 2 80 34 3 3 Worker 3 50 4 1 1 Worker 4 10 0 Worker 5 30 0
|
10.1038/s41598-020-75728-9
|
cc-by
| null | null |
openalex
|
Duckweeds are small, free-floating, morphologically highly reduced organisms belonging to the monocot order Alismatales. They display the most rapid growth among flowering plants, vary ~ 14-fold in genome size and comprise five genera. Spirodela is the phylogenetically oldest genus with only two mainly asexually propagating species: S. polyrhiza (2n = 40; 160 Mbp/1C) and S. intermedia (2n = 36; 160 Mbp/1C). This study combined comparative cytogenetics and de novo genome assembly based on PacBio, Illumina and Oxford Nanopore (ON) reads to obtain the first genome reference for S. intermedia and to compare its genomic features with those of the sister species S. polyrhiza. Both species' genomes revealed little more than 20,000 putative protein-coding genes, very low rDNA copy numbers and a low amount of repetitive sequences, mainly Ty3/gypsy retroelements. The detection of a few new small chromosome rearrangements between both Spirodela species refined the karyotype and the chromosomal sequence assignment for S. intermedia. Duckweeds are the smallest and fastest-growing flowering plants, and are considered as potential aquatic crops, serving for feed, food, fuel and waste water remediation . They comprise 36 largely asexually propagating species within the 5 genera Spirodela, Landoltia, Lemna, Wolffiella and Wolffia With decreasing phylogenetic age duckweed frond sizes decrease from 1.5 cm to less than 1 mm in diameter accompanied by a successive reduction or loss of roots and a nearly 14-fold genome size variation (from 160 to 2203 Mbp) . These features make duckweeds an interesting subject for genome and karyotype evolution studies. So far, no correlation between genome size, chromosome number as well as ribosomal DNA loci was recorded from eleven species representative for five duckweed genera 18 . The genus Spirodela harbors only two species of similar genome size (160 Mbp), S. polyrhiza and S. intermedia. Due to its basal ancestral phylogenetic position, its industrial potential and its small genome, the Greater Duckweed S. polyrhiza was chosen as the first duckweed for whole genome sequencing 21 . By integrating different approaches: cytogenetics, optical mapping (BioNano technique), Hi-C chromatin conformation study, 454, Illumina and Oxford Nanopore sequencing platforms, a high-confidence genome map for S. polyrhiza was established 22, 24 that corrected the errors of previous genome maps 21, . This high-quality genome map provides a source for advanced genomic research regarding repetitive sequences and protein-coding genes, their chromosomal location and evolutionary history in other duckweeds. Moreover, whole-genome duplication (WGD) events and chromosomal rearrangement between duckweed species can potentially be uncovered using the S.Genome assembly for S. intermedia clone 7747 based on the library of PacBio sequence reads. Two rounds of PacBio-sequencing of a 20 kb library of genomic DNA of S. intermedia clone 7747 resulted in 149 Gbp of raw read data. After an initial filtering for potential bacterial contamination, reads of at least 500 nucleotides were assembled using the Canu pipeline v. 1.5 32 . A total of 1,305,064 reads were assembled into 1172 sequence contigs of 147,613,042 nucleotides, corresponding 91.7% of the estimated genome size. All contigs of this draft assembly are covered in median 37.5-fold by raw reads. In a first round of scaffolding, the two genomes of the sister species S. polyrhiza (from clones 9505 and 7498) 23, 25 were used as references to order contigs as described 33 . The resulting scaffolds (N50 = 1,7 Mbp) were super-scaffolded by SSPACE-Longread v.1-1 34 and assigned to the 18 chromosomes of S. intermedia, using 93 S. polyrhiza BACs as landmarks which were cross-hybridized to the S. intermedia chromosomes of clone 8410 29 . In addition to confirmation of the same linkage relationship in clone 7747 (Fig. 1 ), new cytogenetic probes using BACs from the genomic regions of interest were designed for FISH experiments to approve localization of the contigs within the pseudomolecules, to resolve mis-assemblies and/or to confirm new linkages (see below). Furthermore, contiguity of the assembly was confirmed by corrected ON reads of S. intermedia clone 8410 using minimap2 v2.16 35 (see below). After reiterative rounds of manual curation and validation by FISH, in the final genome assembly, 18 scaffolds (N50 = 8.3 Mbp) of in total 131.4 Mbp (82.2% of the estimated genome size) could be assigned to the 18 chromosomes (Table 1 ). Six of them show telomeric sequences at both ends and seven at least on one end (see Fig. 2 ). Most of the shorter and/or repetitive sequences (16.2 Mbp, corresponding 10.1% of the estimated genome size) (N50 = 27.1 Kbp) could not yet be assigned and were considered as additional pseudomolecule "SiUn". Genome assembly for S. intermedia clone 8410 based on Illumina and Oxford Nanopore reads. The ON/Illumina-derived scaffolds for clone 8410 were created using the MaSuRCA assembler and filtered using minimap2 to remove duplicated sequences derived from heterozygous regions. To form pseudomolecules, all 70 remaining scaffolds were corrected and ordered by manual curation using the assembly of clone 7747 as a reference. Finally, all 18 pseudomolecules ended at both sites with telomeric sequences. Merging of both assemblies revealed further small rearrangements between the karyotypes of S. polyrhiza and S. intermedia (see below and Figs. 2 and 3 ). The quality of both S. intermedia genome assemblies was assessed by the BUSCO program v. 3.1.0 36, 37 with Embryophyta dataset 10 including 1375 genes (mostly from land plants). The hybrid assembly of ON/Illumina reads revealed 96.1% of the 1375 searched genes (Table 1 ).
Gene prediction. Based on similarity to nine aquatic and non-aquatic angiosperm reference genomes, including two duckweed species, S. polyrhiza 7498 v3.1 25 and Lemna minor 5500 38 , gene model prediction via Gene Model Mapper-GeMoMa 39 suggested in total 22,245 (Pacbio, clone 7747) or 21,594 (ON/Illumina, clone 8410) protein/RNA-coding genes, some more than predicted for S. polyrhiza 25 (Table S1 ). A total number of 16,162 genes of clone 7747, 16,493 of clone 8410 and 11,327 of S. polyrhiza clone 9509 are coinciding with eggNOGs (Non-supervised Orthologous Groups). Comparing the proportion of eggNOG functional categories between the genomes of the two Spirodela species, the differences were < 1%. Only the category 'Energy production and conversion' , is overrepresented in S. intermedia clone 7747 (4.2 versus 3.1% in clone 8410 and 2.6% in S. polyrhiza) and the category 'Replication, recombination and repair' in clone 8410 (5.3% versus 3.2% in clone 7747 and 2.3% in S. polyrhiza) (Fig. S2 , Table S2 ). Enumeration is as in Hoang & Schubert (2017) 29 and in Table S3 (Short sequences present in S. p., but not chromosomally assigned in S. i. correspond to "SiUn" and are not considered). 13 ChrSi of the 7747 assembly show telomeric sequences at one (*) or both ends (), while all pseudomolecules of the 8410 assembly show them at both ends. New linkages in S. intermedia as revealed by genome assembly and FISH. The PacBio assembly for clone 7747 indicated a new rearrangement involving ChrSp20. This chromosome did not form the entire ChrSi18 as reported 29 , instead it is split into two parts, the largest part corresponding to ChrSi18 and two rather small regions (between ChrSp20 3.72-3.78 and 3.80-3.96 Mbp) transferred to ChrSi16 (42,000-175,000 bp) (Table S3 ). Therefore, the previously not tested BAC 013O04 belonging ChrSp20 was selected for mcFISH experiments. The presence of this BAC sequence in S. polyrhiza ChrSp20 was confirmed by FISH (Fig. 4a ). FISH results on S. intermedia chromosomes (clone 8410: Fig. 4b , and clone 7747: Fig. 4c ) showed that BAC 013O04 (yellow) labeled the same chromosome (ChrSi16) as the BACs belonging to ChrSp15 (red), while the remaining part of ChrSp20 (green) labeled another chromosome pair (ChrSi18). This result confirmed that ChrSp20 became split and a very small part became translocated to ChrSp15, forming ChrSi16 (Fig. 2 ), as inferred by PacBio assembly. Because only 76 kbp of the BAC 013O04 sequence appeared in the ON/Illumina assembly of ChrSi16, this link is not visible in Fig. 3 where the entire chromosome ChrSp20 is represented by the S. intermedia chromosome ChrSi18, based on the assembly results for clone 8410. Furthermore, a piece of ChrSp13 (0.46-0.68 Mpbs), according to the ON assembly for ChrSp13 of S. polyrhiza clone 9509, became integrated into ChrSi02 (5.43-5.66 Mbps) as suggested by the assemblies for both S. intermedia clones (for clone 8410 see Fig. 3 ). However, three BACs from this region (029A10, 028I16 and 037J09, together 649,507 bp) used as FISH probe, labeled ChrSp02 of S. polyrhiza clones 7498 and 9509 (Fig. 5 ) and appear on ChrSp02 also in the Bionano map (CP019095.1) of S. polyrhiza clone 9509 25 as well as in pseudomolecule 1 (corresponding ChrSp02) of S. polyrhiza clone 7498 23 (Table S4 ). This uncovers a hitherto overlooked error in the ON assembly for S. polyrhiza clone 9509 24 . Another new small region, for which no BAC is available, became transferred from ChrSp14 (4.9-5.4 Mbps) to ChrSi04 (9.5-9.9 Mbps) and appeared in both assemblies (for clone 8410 see Fig. 3 ). Although only one BAC from ChrSp16 was tested previously 29 , the assemblies from PacBio and ON reads suggests that the entire ChrSp16 is included in S. intermedia ChrSi11 together with a part of ChrSp10 (Figs. 2, 3 ).
Types, abundance and distribution of repetitive elements. Characterization of S. intermedia repet- itive sequences was first performed by analyzing unassembled Illumina reads from the clone 8410 using the RepeatExplorer pipeline 40 . This analysis served as a control for repeat quantification in the assembled pseudomolecules that may be biased due to the exclusion of satellite DNA or other repeats that are hard to assemble. In addition, the RepeatExplorer output was used to compile a reference database of S. intermedia repeats that was used to annotate the genome assemblies. The RepeatExplorer analysis of 2.4 million paired-end Illumina reads (2.25 × genome coverage) revealed relatively small proportions of highly and moderately repeated sequences in the S. intermedia genome (Table 2 ). The repeats accounted for 20% of the genome, with LTR-retrotransposons representing the most abundant repeat class (14.1% of the genome, with Ty3/gypsy to Ty1/copia ratio of 2.17). Other repeats including LINEs, rDNA and satellite DNA made up only minor genome proportions. Repeat annotations of the genome assemblies using the RepeatExplorer reference database resulted in 18.5% and 20.2% of the 8410 and 7747 sequences marked as repetitive, respectively. These proportions correspond to the estimate obtained for unassembled reads (20.0%), suggesting that repeats were not significantly depleted in the final assemblies. Inspection of individual repeat categories revealed that partial depletion resulting in smaller proportions in the assembled genomes occurred for satellite repeats in both clones and for rDNA in the clone 7747 (Table 2 ). To obtain an alternative estimate of repeat proportions, we also analyzed both assemblies using RepeatScout 41 that performs de-novo identification of repetitive elements based on the high frequency k-mers. This program estimated total repeat proportions of 23.1% (8410) and 25.6% (7747), most likely due to its better sensitivity for low-copy repeats (Table 2 ). An interesting observation was the relatively high abundance of simple-sequence repeats, especially the microsatellite motif (AG) n , that was revealed by Tandem Repeats Finder 42 analysis within the assembled genomes and the Illumina reads. About 37,000 loci of (AG) n with an average length of 38 bp were detected in 8410, making up 1% of the assembly (the same proportion and characteristics were found for (AG) n in the 7747 assembly). These dispersed simple repeat loci appeared in the S. intermedia genome with an average frequency of one per 3.7 kb. Characterization of rDNA loci. Chromosome analysis of S. intermedia by FISH using 5S and (18S + 26S) rDNA probes, detected one major locus of 5S rDNA on ChrSi15 (corresponding to ChrSp13) (Fig. S3a ) and a major locus of 45S rDNA on ChrSi01 (corresponding to ChrSp01) (Fig. S3b ). The chromosome-scale assembly and ON reads for Si8410 confirmed the presence of a 5S rDNA locus on ChrSi15, and revealed an additional locus on ChrSi14. The extra-long ON reads showed that the locus on ChrSi15 contains a cluster of thirty-one 5S rDNA repeats, whereas the locus on ChrSi14 is composed of seven and 13 5S rDNA repeats interrupted by two 6 kb long repeats of another sequence (Fig. 6b ). The chromosome assembly, based on PacBio sequences, showed similar arrangement of 5S rDNA in Si7747 with slightly different copy numbers in each of the loci. Quantitative PCR estimation of 5S rDNA copy number in Si7747 and Si8410 genomes supports this data with 57 ± 10 copies of 5S rRNA genes for Si8410 and, 70 ± 20 copies for Si7747. Sequence alignments suggested that the 5SrDNA units of the two loci contain slightly different non-transcribed spacers (NTS). This was confirmed by sequencing of the 5S rDNA repeats, amplified from genomic DNA of clones 8410 and 7747. Analysis of individual 5S rDNA clones of Si8410 and Si7747 identified the 119 bp long 5S rRNA genes with 100% identity to the previously sequenced 5S rRNA gene of S. polyrhiza 43 , and two variants of the NTS for each ecotype (Fig. S4 ), with variant 8410-1 corresponding to 5S rDNA locus on chromosome ChrSi15 and the variant 8410-2 corresponding to the locus on chromosome ChrSi14 (Fig. 6a ). In agreement with the FISH data, the genome assembly revealed a 45S rDNA locus in a distal position on ChrSi01 for both clones, Si7747 and Si8410. Extra-long ON reads confirmed this locus for Si8410, about 83 kb upstream of one ChrSi01 chromosome telomere. Moreover, ON reads revealed an additional cluster of 45S rDNA repeats about 8 kb upstream the telomere on ChrSi06. Unlike the situation with 5S rDNA loci, there are no readthrough sequences containing the whole 45S rDNA loci. Therefore, it is not possible to determine exactly how many of the 93 ± 25 45S rDNA copies estimated by qPCR for Si8410, are located in the minor locus.
Discussion Chromosome rearrangements between the two Spirodela species. Previously, chromosome homeology and rearrangements between S. polyrhiza and S. intermedia were uncovered by comparative serial multicolor cross-hybridization to the S. intermedia clone 8410 29 . Here, we applied the same approach, using informative BACs out of the 96 ones anchored in the S. polyrhiza genome, and found the same chromosomal rearrangements in S. intermedia clone 7747 as reported for clone 8410 29 . Reiterative rounds of genome assembly and validation by FISH revealed a new linkage compared to the S. polyrhiza genome, in addition to the eight translocations detected previously 29 . This new linkage involves ChrSi16, which received small parts from one end of ChrSp20 (Fig. 2 ). A small piece, apparently transferred from one end of ChrSp13 to ChrSi02, turned out to be an error within the previous ON assembly for ChrSp13 of clone 9509 24 . Another small region, transferred from ChrSp14 to ChrSi04 was not studied by FISH, but it appeared in both assemblies. The S. intermedia chromosomes ChrSi03, 04, 06, 07, 09 and 11, previously found to be involved in evolutionary rearrangements, correspond in total to 10 S. polyrhiza chromosomes (Figs. 1, 2, 3 and 29 ). Now, at least two more rearrangements (in addition to the previously postulated one inversion and eight translocations) have to be assumed if the S. polyrhiza karyotype is more similar to that of the ancestor. Alternatively, eight (instead of six) translocations (involving ChrSi03, 04, 06, 07, 08, 11, 12, 16, 18), and one fission (ChrSi09) 29 were required if the S. intermedia karyotype is the more ancestral one. Additionally, we found a positional change of the smaller 5S rDNA locus to ChrSi14 (instead of ChrSi07 which harbors sequences adjacent to the minor 5S rDNA locus on ChrSp06). The increased number of rearrangements refined our knowledge about karyotype evolution between S. polyrhiza and S. intermedia and made the corresponding genome assembly for both S. intermedia clones robust by independent confirmation. These results demonstrate the benefit of our novel approach, combining comparative cytogenomics and hybrid sequencing technologies, especially for species for which genetic maps are not available. Genome assembly for both S. intermedia clones. The assembly of PacBio reads for clone 7747 is more fragmented than the assembly of ON/Illumina reads for clone 8410. It displays a higher percentage of ambiguous bases (0.003%) and a lower coverage (Table 1 ). Several draft contigs representing contamination from plastids, mitochondria, and a virus (Human alpha herpesvirus 1 strain) were detected which show largely increased coverages of 2925-, 17,126-and 73-fold respectively. These overrepresented sequences caused a diminished overall coverage in both assemblies. Canu performs best with more than 50X coverage, while lower values might decrease the contiguity of an assembly 32 . Comparing read length N50 of the two S. intermedia clones, the ON library of clone 8410 (16,322 kb) yielded a larger amount of long reads than clone 7747 with an N50 of 9879 kb. As a consequence, longer repeats may not be sequenced completely, leading to a more fragmented assembly 44 , as in case of clone 7747. In summary, the PacBio read assembly of S.intermedia 7747 provides continuous pseudomolecules but these might comprise ambiguous and low coverage nucleotide stretches, and possibly missing sequences, as described for other plant species 45 . Despite the lower library quality for clone 7747, the non-hybrid PacBio assembly shows (as expected 46 ) good results for the scaffold N50 (8.3 Mbp similar to 9.25 of the ON/Illumina assembly, Table 1 ), depicting the synergy of cytogenetics and long read sequencing technologies. The distribution of functional eggNOG groups within the S. intermedia clones and S. polyrhiza is largely balanced, indicating a high similarity in overall functional gene content between both species which amounts with ~ 20,000 genes, a rather low number compared to most land plants. The BUSCO results reflect the increased fragmentation of the 7747 assembly by an increased number of fragmented and missing genes (~ 20% in total). With 3.0% of fragmented and 3.9% of missing candidate genes, the ON based assembly of clone 8410 exhibits a good representation of the expected gene content. It has to be noted, that the embryophyta set of orthologue sequences (mostly from land plants) might not be optimal for aquatic plants, because unnecessary genes might have been lost during evolutionary organ reduction and aquatic life style of duckweeds 21 . Repetitive elements. S. intermedia revealed a rather low proportion of detected repeats (~ 25%), in comparison with other genomes of similar size, such as that of A. thaliana (157 Mbp/1C; 32% 47 ), or the even smaller genome of Genlisea nigrocaulis (86 Mbp/1C) with 15.9% of total repeats 48 . Similar repeat proportions were reported by Wang et al. ( 2014 ) 21 for S. polyrhiza, however, later Michael et al. (2017) 25 reported a higher proportion of mobile elements (25%) for that species. Possible explanations might be: (1) There is a difference in repeat content between S. polyrhiza and S. intermedia; this seems less likely, because of the close relationship and same genome size of both species. (2) The assembly by Michael et al. 25 is more complete; this is probably not the case, because we found a lower content of mobile elements also in unassembled reads. (3) Their methods including structure-based detection revealed low copy elements (one or a few copies/C) which are not captured by our clustering and similarity-based searches. (4) Michael et al. 25 described a ratio of soloLTRs to complete elements of 8 to 1. Because our tools did not determine soloLTRs, the repeat content for S. intermedia might be underestimated. A high proportion of soloLTRs suggests that the genome size of Spirodela decreased by deletion-biased DNA double-strand repair 49 via the single-strand annealing pathway since its separation from the other duckweed lineages. Remarkable is the high abundance of SSR repeats, especially that of dispersed (AG) n microsatellite arrays, which in rice contribute to regulate gene expression by binding transcription factors 50 . Such arrays were reported also for S. polyrhiza, where they "severely impeded elongation during sequence assembly" 21 . The unusually low copy number of both 45S and 5S rDNA repeats revealed in the genome of S. polyrhiza 24, 25 , inspired curiosity about the number and arrangement of those genes in related S. intermedia. The qPCR-based estimation showed ~ 93 copies of the 25S rRNA genes for both Si8410 and Si7747 genomes, a number very close to the estimate for S. polyrhiza 25 . Unlike S. polyrhiza, where the 45S rDNA was shown to locate in a single chromosome locus, our deep coverage ON sequencing of Si8410 revealed at least two 45S rDNA loci, located at ~ 83 kb upstream of the telomere at SiChr01, and at ~ 8 kb upstream of the telomere at SiChr06. The fact that FISH revealed only one signal on SiChr01, suggests that this location is the major 45S rDNA locus with the majority of the ~ 93 gene repeats. However, the exact 45S rDNA copy number distribution between the two loci remains unclear, because no read-through sequences are available. For the 5S rDNA, a range of generated ON read-through sequences, revealed two distinct loci, one containing a cluster of 31 repeats on SiChr15 which has been also visualized by FISH, and a second split locus on SiChr14 containing two clusters with 7 and 13 repeats, separated by ~ 13 kb long region of non-rDNA sequence (Fig. 6b ). While the general arrangement of the 5S rRNA genes in S. intermedia resembles that of the S. polyrhiza, the total 5S rDNA copy number in S. intermedia is even lower, 51 vs 73 in S. polyrhiza 24 . This is the smallest number reported so far for any plant species. Usually, the copy number of 5S RNA genes in land plants varies from 2000 to 75,000 51 . Therefore, our findings for S. polyrhiza and S. intermedia proclaim that the small copy number of rDNA is a unique phenomenon of the genus Spirodela.
Material and methods Plant material. Spirodela intermedia W. Koch (accessions 8410 from Panama City and 7747 from Lima, Peru) were obtained from Elias Landolt's collection via Klaus Appenroth, University of Jena and Rutgers Duckweed Stock Cooperative (New Jersey, USA). The fronds were grown in liquid nutrient medium 52 under 16 h white light of 100 μmol m -2 s -1 at 24 C. Genomic DNA isolation. Genomic DNA of S. intermedia (clone 7747) was extracted from fresh fronds using the DNeasy Plant Mini Kit (Qiagen) for PacBio sequencing. High molecular weight DNA of S. intermedia (clone 8410) was isolated for Oxford Nanopore and Illumina sequencing as follows: the plants were kept three days in the darkness. After harvesting, 10 g of the fronds were used for DNA isolation from purified nuclei according to the protocol of Vondrak et al. 53 , with two minor modifications: the centrifugation of nuclei was performed at 650 × g instead of 200 × g due to their small size, and 2 × CTAB isolation buffer was supplemented with 2% PVP-360 (polyvinylpyrrolidone, avg. molecular weight 360,000). Genome sequencing. PacBio. After shearing of genomic DNA (S. intermedia clone 7747), a size-selected 20 kb library was sequenced on the Pacific Biosciences RS II platform (GATC Biotech, Konstanz, Germany) combining the P6-C4 polymerase-chemistry and 240 min of movie duration. Two rounds of sequencing resulted in 149 Gb of raw read data. Oxford nanopore. The sequencing libraries were prepared from 3 μg of purified HMW DNA using a Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) as described by Vondrak et al. 53 . Briefly, the DNA was treated with 2 μl of NEBNext FFPE DNA Repair Mix and 3 μl of NEBNext Ultra II End-prep enzyme mix in a 60 μl volume that also included 3.5 μl of FFPE and 3.5 μl of End-prep reaction buffers (New England Biolabs). The reaction was performed at 20 C for 5 min and 65 C for 5 min. Then, the DNA was purified using a 0.4 × volume of AMPure XP beads (Beckman Coulter). Because long DNA fragments caused clumping of the beads and were difficult to detach, the elution was performed with 3 mM TRIS-HCl (pH 8.5) and was extended up to 40 min. Subsequent steps including adapter ligation using NEBNext Quick T4 DNA Ligase and library preparation for sequencing were performed as recommended. The whole library was loaded onto FLO-MIN106 R9.4 flow cell and sequenced on MinION instrument until the number of active pores dropped below 40 (21-24 h). Basecalling of the raw reads was done using Albacore 2.3.3 (Oxford Nanopore Technologies). Illumina. Illumina sequencing was performed by Admera Health, LLC (South Plainfield, NJ, USA) using a KAPA DNA Library kit (Roche) and resulted in 130 million paired-end reads (2 × 150 nt).
Sequence assembly and pseudomolecule construction. After an initial filtering for potential bacterial contamination (blastn against microbial NCBI refseq database from Aug 2017) and minimum read length (500 nucleotides), PacBio reads were assembled using the Canu pipeline v. 1.5 32 (options: 'genomeSize = 160 m, correctedErrorRate = 0.105′) consisting of the following steps: (1) Trimming, error correction and contig construction: Reads were corrected and trimmed by comparing overlaps. A minimum length of 500 nucleotides and a maximum error rate of 10.5% was chosen for extending a contig. Only reads consisting of more than 1000 nucleotides in length were considered in this step. Afterwards, the corrected reads were trimmed to improve overall read quality by using overlap information to detect high confidence regions. Contigs of insufficient read coverage and/or containing 'noisy' sequence were categorized as 'unsupported regions' and divided at weak sequence positions into subcontigs with higher support. After further contig construction on the basis of overlaps, a consensus sequence was built by removing the remaining sequencing errors to raise the overall assembly quality. (
2) Scaffolding and gap filling In a first round of scaffolding, the two genomes of the sister species S. polyrhiza (from clones 9505 and 7498) 23, 25 were used as references for Mauve Genome Aligner v20150522 33 to order contigs. Scaffolding was performed by SSPACE-Longread v.1-1 34 (default options). The resulting scaffold assembly was used for the super-scaffolding approach. For this aim, contigs were assigned to 18 putative pseudomolecules (corresponding to the 18 S. intermedia chromosomes) using the information of cross-FISH of 93 S. polyrhiza BACs on the chromosomes of S. intermedia clone 8410 29 . New cytogenetic probes using BACs from the genomic regions of interest were designed for FISH experiments to approve localization of the contigs within the pseudomolecules and to resolve mis-assemblies. Additionally, bacterial contamination was filtered as described previously. The quality of both S. intermedia genome assemblies was assessed by the BUSCO program 36, 37 with the Embryophyta odb10 dataset comprising 1375 conserved genes. The ONT/Illumina assembly for clone 8410 was performed using MaSuRCA 54 . In the first step, super-reads were assembled from 68 million Illumina 2 × 151 nt paired-end reads (128-fold coverage). Subsequently, 307,111 nanopore reads 10,000-425,377 nt in length (total length 9,963,752,003) and representing a 62-fold genome coverage, were used in scaffolding step. The resulting MaSuRCA assembly consisted of 386 scaffolds (N50 408,333 bp, total length 191,862,084 bp). Scaffolds were assigned/ordered into pseudomolecules using the 7747 as reference for a Mauve Genome alignment 33 . Additionally, all the formation of super-scaffolds was accompanied by manual curation steps based on FISH results. Gene prediction and functional annotation. Gene finding was carried out using Gene Model Mapper (GeMoMa)-a similarity-based gene prediction program 39 ('GeMoMa-1.6.1.jar CLI GeMoMaPipeline t = g = a = Extractor. p = true AnnotationFinalizer.r = SIMPLE AnnotationFinalizer.p = '). Gene models were predicted by combining the predictions based on the genome data of the neighbor species S. polyrhiza and eight additional reference organisms (S. polyrhiza 7498 v3.1 23 , Lemna minor 5500 38 , Arabidopsis thaliana TAIR10, Ananas comosus (Phytozome internal code 321), Brachypodium distachyon (Phytozome internal code 314), Nelumbo nucifera 1.1 (GenBank assembly accession: GCF_000365185.1), Panicum hallii v3.1 (GenBank assembly accession: GCF_002211085.1), Oryza sativa IRGSP v1.0.38 (GenBank assembly accession: GCA_001433935.1), Zostera marina v2.1 (GenBank assembly accession: GCA_001185155.1)). Noncoding RNAs were determined using RNAmmer v1.2 55 ('rnammer -S euk -m tsu,ssu,lsu -h -f ') and tRNAscan-SE-2.0 56 ('tRNAscan-SE -B -o -b -thread 10′). Predicted tRNAs were filtered for features overlapping with protein coding exons ('bedtools intersect -b -a -wa -wb -f 0.8 -r'). Functional annotations and GO terms were assigned using Interproscan 5 v 5.26-65.0 57 ('interproscan.sh -dp -input -seqtype p -f tsv,html,gff3 -applications TIGRFAM, PfamA, SMART, SUPERFAMILY -pathways -goterms'). COGs (clusters of orthologous groups) were computed using eggnog-mapper 58 online tool ('Taxonomic background: Viridiplantae, default options') based on eggNOG 4.5 orthology data 59 . Comparative genomics and visualization. Best bi-directional hits (BBHs) were identified using BLASTN 2.2.31 + ('-evalue 1e-5 -out outfmt 6′). Additional filtering for length and percent identity was applied afterwards (minimum length: 100 nt; minimum identity: 80%). Annotation of gene features and repetitive elements in S. polyrhiza 9505 was taken from Michael et al., 2017 25 . Both, length of selected repeat features and gene density were computed across a 0.5 Mbp window using the bedops suite 60 . Feature annotation of each annotated repeats and genes as well as chromosome sizes have been converted to bed format using 'gtf2bed' . From these files the 0.5 Mbp windows across each chromosome have been calculated with bedops -chop. Finally, the counts of gene and repeat features within each chromosome has been determined by bedmap -count. Synteny was determined using minimap2 ('minimap2 -x') 35 between S. polyrhiza and S. intermedia. 8410. Overlapping intervals have been merged by bedtools 61 merge (default options). Links shorter than 10.000 have been excluded from the analysis. To compare the two Spirodela genomes, synteny between pseudomolecules, gene and repeat distribution have been plotted using Circos tool v0.67-1 62 . Repeat annotation and analysis. Repeat analysis in unassembled Illumina reads from the clone 8410 was performed using similarity-based clustering implemented in the RepeatExplorer pipeline 40 . The pipeline was run with modified settings in order to increase its sensitivity towards divergent repeats. The similarity search step was done with BLASTN instead of mgblast and the similarity threshold was lowered from 90 to 80% identity. A total of 2.4 million of 150 nt paired-end reads were used as an input, and repeats were annotated in clusters representing at least 0.005% of the input reads. Repeat annotation in the assembled pseudomolecules was performed using several alternative approaches. PROFREP and DANTE modules available at the public RepeatExplorer server (https ://repea texpl orer-elixi r.cerit -sc.cz/) were used to annotate repeats based on similarities to the reference database compiled from the repeat clustering analysis described above, and to the REXdb database of conserved protein domains of mobile elements 63 , respectively. Additionally, repetitive sequences in the assemblies were annotated using RepeatScout v. 1.0.5 41 . First, a library of repeats based on frequent k-mers was created using default parameters. Repeats with a frequency below 10 copies were then removed from the library. The resulting library was then used for annotating genome assemblies using RepeatMasker (https ://www.repea tmask er.org). Identification and quantification of microsatellites (simple sequence repeats) was done by means of Tandem Repeat Finder 42 using the settings "trf input_file 2 5 7 80 10 25 25 -f " and parsing the output using TRAP 64 . Molecular characterization of rDNA. The estimation of 25S and 5S rRNA gene copies was carried out by qPCR, relating the rates of sample DNA amplification to the standard curve. The standard curve was constructed based on the amplification reads of dilution series of a reference plasmid, containing part of the actin gene (single copy in Spirodela polyrhiza, 9509), a whole PCR amplified 5S rDNA unit of S. polyrhiza and a part of the 25S gene amplified from genomic DNA of S. polyrhiza, 9509, using primers with internal restriction sites for XbaI and EcoRI. The rDNA copy number was determined in qPCR reactions prepared with the UltraSybr Mixture (CWBio, Taizhou, China), run on the CFX Connect Real-Time detection system (Bio-Rad, Hercules, USA). For quantification of the 25S rDNA we used the 5′-TCC CAC TGT CCC TGT CTA CT and 5′-CCC ACT TAT CCT ACA CCT CT primers, and for the 5S rDNA the set of primers was: 5′-GGG TGC GAT CAT ACC AGC AC and 5′-GGG TGC AAC ACG AGG ACT TC. The samples and tenfold dilution series of the reference plasmid were assayed in the same run. The quality of products was checked by thermal denaturation cycle. Only the experiments providing a single peak were considered. Three technical replicates were performed for each sample. The obtained data were analyzed using the program BIO-RAD CFX Manager 3.1 (Hercules, USA) and Microsoft Excel 2016 software. For sequencing, the 5S rDNA genes were amplified from genomic DNA by PCR using 5S rRNA gene-specific primers DW-5S-F: CTT GGG CGA GAG TAG TAC TAGG and DW-5S-R: CAC GCT TAA CTT CGG AGT TCTG, purified by gel electrophoresis and cloned into the vector pMD19 (Takara, Dalian, China). The obtained sequences were analyzed using the "Online Analysis Tools" package (https ://molbi ol-tools .ca).
Mitotic chromosome preparation, probe preparation and FISH. Spreading of mitotic chromosomes was carried out according to Hoang 29 . 5S rDNA, 18S and 26S rDNA probes were generated from S. polyrhiza and from S. intermedia genomic DNA each by using designed primer pairs 16, 21, 65, 66 as described 24 . Ribosomal DNA, A. thaliana type telomere and S. polyrhiza BAC probes were labeled with Cy3-dUTP (GE Healthcare Life Science), Alexa Fluor 488-5-dUTP, Texas red-12-dUTP, biotin-dUTP or digoxigenin-dUTP (Life Technologies) and precipitated as described 29 . Figure 1 . 1 Figure 1. Six linkages due to chromosome rearrangements between S. polyrhiza and S. intermedia (clone 8410) are also present in the sequenced clone 7747. (a) ChrSp05-ChrSp06 = ChrSi06; (b) ChrSp08-ChrSp18 = ChrSi09; (c) ChrSp03-ChrSp17 = ChrSi04; (d) ChrSp10-ChrSp16 = ChrSi11; (e) ChrSp06-ChrSp07-ChrSp14 = ChrSi07; (f) ChrSp03-ChrSp06-ChrSp14 = ChrSi03. See also Fig. 3. Scale bars = 5 μm.
Figure 2 . 2 Figure 2. Rearrangements between S. polyrhiza (n = 20) and S. intermedia (n = 18), confirmed for chromosomes of clones 8410 and 7747. Enframed: newly found rearrangement. Red boxes: present only in S. polyrhiza; green boxes: sequences present only in S. intermedia. Scale bar = 3 Mbp (based on PacBio assembly for clone 7747).Enumeration is as in Hoang & Schubert (2017) 29 and in TableS3(Short sequences present in S. p., but not chromosomally assigned in S. i. correspond to "SiUn" and are not considered). 13 ChrSi of the 7747 assembly show telomeric sequences at one (*) or both ends (), while all pseudomolecules of the 8410 assembly show them at both ends.
Figure 3 . 3 Figure 3. Circos plot of genomes of S. polyrhiza 9505 (orange) and S. intermedia 8410 (blue). (A) tracks representing the size of the pseudomolecules with a corresponding scale in 1 Mbp steps, with highlights every 5 Mbps, (B) total length of repeat features (in kbps) (C) gene density and (D) pairwise sequence synteny. The synteny link between gi|13 and Si02 is based on an error in the ON assembly for clone 9509 (see Fig. 5). The region in question actually belongs to gi|2 as does the remaining part of Si02. Gene and repeat density are plotted in 0.5 Mbps bins. Data for S. polyrhiza 9505 are from Michael et al. 2017 25 .
Figure 4 . 4 Figure 4. New rearrangement between ChrSp 20 and ChrSp 15 in S. intermedia (a) The newly tested BAC 013O04 (yellow) belongs to ChrSp 20 (green) in S. polyrhiza; (b,c) BAC 013O04 was translocated to ChrSp 15 (red) forming ChrSi 16 in S. intermedia clones 8410 and 7747. Scale bars = 5 μm.
Figure 5 . 5 Figure 5. Evidence for ON mis-assembly of chromosome 13 of S. polyrhiza 9509. The newly tested BACs 029A10, 028I16 and 037J09 (yellow) belong to ChrSp 02 (red) of S. polyrhiza clone 9509 (upper panel) and clone 7498 (lower panel), not to ChrSp13. Scale bars = 5 μm.
Figure 6 . 6 Figure 6. Schematic representation of rDNA loci in S. intermedia 8410. (a) Depiction of 45S rDNA loci at chromosomes ChrSi01 and ChrSi06; Tel = telomere. (b) Depiction of 5S rDNA loci at chromosome ChrSi15, and ChrSi14. The ChrSi15 locus contains 31 5S rDNA units with NTS of type-1; the ChrSi14 locus is composed of two clusters of 7 and 13 5S rDNA units of type-2 NTS, separated by a doubled 6 kb sequence of unknown function.
Table 1 . 1 Assembly statistics of S. intermedia clones. Sequencing technique PacBio (clone 7747) ON/Illumina (clone 8410) Input data Read coverage a 37.6× 191× Chromosome number 18 Genome physical size b ~ 160 Mbp Assembly statistics and gene prediction Assembly length (Mbp) 147.6 136.6 Number of pseudomolecules 18 Number of contigs 584 86 Number of scaffolds 420 70 Number of assigned scaffolds 18 pseudomolecules (featuring 63 contigs) ~ 131.4 Mbp total length 18 pseudomolecules (featuring 34 contigs) ~ 134 Mbp total length Largest scaffold length (Mbp) 12.5 (Si09) 13.4 (Si09) N50 scaffold length (Mbp) 8.3 9.25 G + C content (%) 41.6 42.0 Number of predicted gene models 22,245 21,594 Completeness of gene prediction (BUSCO) c Complete genes (C) 1097 (79.8%) 1280 (93.1%) Complete and single-copy (S) 1085 (78.9%) 1266 (92.1%) Complete and duplicated (D) 12 (0.9%) 14 (1.0%) Fragmented genes (F) 131 (9.5%) 41 (3.0%) Missing genes (M) 147 (10.7%) 54 (3.9%) Total number of BUSCO genes used 1375 a Based on genome size measurements by FCM. b Measured by FCM. c Reference database odb10.
Table 2 . 2 Repeat proportions [%] estimated for unassembled sequence reads and genome assemblies of S. intermedia clones 8410 and 7747. 8410 8410 7747 Repeat Illumina reads Assembly Assembly Ty3/gypsy Athila 7.44 7.23 8.57 CRM 1.09 1.02 1.13 Reina 0.36 0.32 0.30 Galadriel 0.05 0.04 0.05 Tekay 0.02 0.00 0.00 Ty1/copia Ale 1.87 1.78 1.79 Ivana 0.94 0.91 0.97 Tork 0.69 0.71 0.70 Ikeros 0.40 0.37 0.39 Unclassified 0.23 0.21 0.27 LTR unclass 1.01 0.93 1.08 LINE 0.37 0.42 0.49 Satellite 0.17 0.06 0.09 Microsat. (GA)n 0.67 1.02 0.98 rDNA 0.47 0.34 0.06 Unclassified 4.21 3.11 3.35 Total 20.00 18.49 20.22 RepeatScout n.a 23.11 25.58
Scientific Reports | (2020) 10:19230 | https://doi.org/10.1038/s41598-020-75728-9
|
10.7554/elife.15092
|
cc-by
| null | null |
openalex
|
Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite bhydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of b-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of b-hydroxybutyrate. Electrophysiological measurements indicate that bhydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.Introduction It has been traditionally thought that physical activity and the processes of learning and memory formation are independent and carried out by different organ systems. However, from an evolutionary perspective, these processes needed to be tightly intertwined to ensure the survival of animal species. Indeed, physical effort usually occurred in response to an imminent danger. Responding to that danger not only required running, but also necessitated better functioning of the brain through increased plasticity in order to adapt to new sources of stress, to learn to avoid dangers or better respond to them, and to map surroundings and learn the locations of hazards (Noakes and Spedding, 2012) All of these responses require improved memory. Physical exercise produces many benefits in the brain that enhance cognitive function, blood flow and resistance to injury. One mechanism to account for the changes in brain plasticity is through the action of growth factors (Cotman et al 2007) . A major contributor to the processes of learning and memory formation involves brain derived neurotrophic factor (BDNF) signaling pathways. It has been known for over two decades that physical activity or neuronal activity markedly enhances Bdnf gene expression in the brain (Isackson et al., 1991; Neeper et al., 1995) and that this increase in BDNF protein leads to activation of signaling pathways that result in exercise-dependent enhanced learning and memory formation (Vaynman et al., 2004) . Though these results are widely recognized, it is important to note that very little is known about the molecular mechanisms that link exercise and Bdnf expression. Regulation of Bdnf expression occurs by many means, but how exercise influences the expression of trophic factors is not understood. In this paper, we are interested in understanding how physical exercise induces Bdnf gene expression. This is a significant question, since cognitive ability and synaptic plasticity are influenced by the levels of BDNF (Lu et al., 2013; Park and Poo, 2013; Vaynman et al., 2004) and BDNF signaling is reduced in many neurodegenerative and psychiatric diseases (Autry and Monteggia, 2012; Zuccato and Cattaneo, 2009) . During development, BDNF is required for the survival of specific neuronal populations and it participates in axonal and dendritic growth and synaptogenesis (Alsina et al., 2001; Bibel and Barde, 2000) . A number of studies have indicated that decreased levels of BDNF are associated with depression and become enhanced following antidepressant treatment (Duman and Monteggia, 2006; Martinowich et al., 2007) . Moreover, exercise frequently leads to an increase in BDNF in the central nervous system to promote improvement in cognitive ability and depressive-like behavior (Marais et al., 2009; Russo-Neustadt et al., 2000) . Indeed, physical activity has been shown to have anti-depressant effects and to improve outcomes in animal models and for patients with eLife digest Exercise is not only good for our physical health but it benefits our mental health and abilities too. Physical exercise can affect how much of certain proteins are made in the brain. In particular, the levels of a protein called brain derived neurotrophic factor (or BDNF for short) increase after exercise. BDNF has already been shown to enhance mental abilities at the same time as acting against anxiety and depression in mice, and might act in similar way in humans. Nevertheless, it is currently not clear how exercise increases the production of BDNF by cells in the brain. Sleiman et al. have now investigated this question by comparing mice that were allowed to use a running wheel for 30 days with control mice that did not exercise. The comparison showed that the exercising mice had higher levels of BDNF in their brains than the control mice, which confirms the results of previous studies. Next, biochemical experiments showed that this change occurred when enzymes known as histone deacetylases stopped inhibiting the production of BDNF. Therefore Sleiman et al. hypothesised that exercise might produce a chemical that itself inhibits the histone deacetylases. Indeed, the exercising mice produced more of a molecule called b-hydroxybutyrate in their livers, which travels through the blood into the brain where it could inhibit histone deacetylases. Further experiments showed that injecting b-hydroxybutyrate directly into the brains of mice led to increase in BDNF. These new findings reveal with molecular detail one way in which exercise can affect the expression of proteins in the brain. This new understanding may provide ideas for new therapies to treat psychiatric diseases, such as depression, and neurodegenerative disorders, such as Alzheimer's disease. (Frazzitta et al., 2014) or Alzheimer's disease (Smith et al., 2014) . As a result, by understanding the molecular mechanisms by which exercise induces Bdnf expression, we aim to harness the therapeutic potential of physical exercise and eventually identify novel therapeutic targets for both psychiatric and neurodegenerative diseases. In animal models, exercise induces Bdnf mRNA expression in multiple brain regions (Cotman et al., 2007) , most prominently in the hippocampus. BDNF production provides trophic support and increases in synaptogenesis and dendritic and axonal branching and spine turnover. Blocking BDNF signaling attenuates the exercise-induced improvement of spatial learning tasks (Vaynman et al., 2004) , as well as the exercise-induced expression of synaptic proteins (Vaynman et al., 2006) . However, how BDNF is selectively increased after physical activity-dependent changes in the nervous system is not well understood. One mechanism that has been proposed is that exercise induces Bdnf expression through the induction of expression of Fndc5 (Wrann et al., 2013) , a PGC-1a-dependent myokine. This hypothesis proposes that the FNDC5 protein is cleaved into a small circulating protein called irisin, which has been associated with the browning of fat (Bostrom et al., 2012) . However, there are contradictory reports about whether Fndc5 is translated and expressed at high levels after exercise and whether irisin is produced and found in blood (Albrecht et al., 2015; Jedrychowski et al., 2015) . This raises questions about how and whether a myokine can be responsible for the induction of Bdnf gene regulation. A second hypothesis is that exercise may induce BDNF levels by altering the epigenetic landmarks of the Bdnf promoters (Guan et al., 2009; Koppel and Timmusk, 2013) . Because exercise induces metabolic changes and because epigenetics lies at the interfaces between the environment and changes in gene expression, it is conceivable that an endogenous molecule is produced after exercise, which can serve as a metabolite as well as a regulator of Bdnf transcription. In this paper, we provide a mechanism demonstrating that exercise induces the accumulation of a ketone body (D-b-hydroxybutyrate or DBHB) in the hippocampus, where it serves both as an energy source and an inhibitor of class I histone deacetylases (HDACs) to specifically induce BDNF expression.
Results
Exercise induces Bdnf expression in the hippocampus To assess how exercise enhances Bdnf gene expression, we established a voluntary running protocol for mice (4 weeks of age), which has been previously shown to mediate increases in BDNF (Marlatt et al., 2012) . Mice were individually housed in cages and divided into two groups: control and exercise. Each mouse in the exercise group was provided with a running wheel, whereas the control group mice were not. Mice were not forced to run on the wheels, but rather were allowed to run voluntarily. Long term running for up to 30 days was monitored (Figure 1A ) for distance and time. We chose this exercise model because of its voluntary aspect. Stress has been reported to decrease BDNF levels (Murakami et al., 2005) . For that reason, we avoided any handling or stress induction in the mice. Animals were sacrificed and tissues were collected for analysis. To determine the effects of voluntary running in the hippocampus, we initially focused our study on selective promoters from the Bdnf gene, which has a complex structure containing multiple promoters that generate many transcripts with a common coding exon (Pruunsild et al., 2011) . Promoter I (pI), a neuronal activity dependent promoter (Tabuchi et al., 2002) and other nearby promoters (pIIA, pIIB, pIV) (Pruunsild et al., 2011) , were also assessed for mRNA expression. Our results showed that voluntary exercise for 4 weeks significantly induced BDNF promoter I and II expression in the Figure 1 continued (B) Voluntary exercise for 4 weeks significantly induces Bdnf promoter I expression in the hippocampus as measured by real-time RTPCR. The number of animal used for each group (control and exercise) is 10. *p<0.05 as measured by unpaired t-test. (C) Western blot analysis depicting the increase in mature BDNF protein levels in the hippocampus of exercise animals as compare to wild type. In this representative image, the BDNF levels from 2 control hippocampal lysates and 3 exercise hippocampal lysates are depicted. This experiment was replicated from additional 3 different animals in each group. (D) Quantification of the BDNF western blot. DOI: 10.7554/eLife.15092.003 hippocampus (Figure 1B and data not shown). Western blot analysis of the hippocampus of animals after voluntary running showed a significant increase of mature BDNF protein levels, as compared to control mice (Figure 1C and D ).
Exercise induces changes in class I HDAC expression and binding to the hippocampal Bdnf promoter One epigenetic mechanism that has been proposed for inducing Bdnf gene expression is histone acetylation (Koppel and Timmusk, 2013) . A number of studies have reported that blocking HDACs enhances memory formation and the expression of synaptic plasticity genes, such as Creb, Bdnf and CamkII (Guan et al., 2009; Koppel and Timmusk, 2013) . HDACs catalyze the deacetylation of histones and are divided into four classes. Class I (HDAC1, HDAC2, HDAC3 and HDAC8), II and IV are zinc dependent enzymes and are represented by at least 11 different proteins, whereas class III includes the sirtuins (Haberland et al., 2009) . We have found that the general class I/IIb HDAC inhibitor, SAHA (vorinostat), a clinically approved anticancer agent and memory enhancer (Guan et al., 2009) and TSA (trichostatin A), a broad-spectrum inhibitor, were effective in inducing Bdnf mRNA in primary cortical neuron cultures (Figure 2A ). More specific HDAC inhibitors, such as CI-994 and MS-275, that target class I HDAC members were also capable of elevating Bdnf transcripts (Figure 2B ), whereas a non-active analog of CI-994, BRD4097 (manuscript submitted), did not give a response. While these results implicate class I HDAC inhibition in the regulation of synaptic plasticity genes, such as Bdnf, it is not clear how exercise is translated into increases in BDNF. Interestingly, we found that exercise reduces Hdac2 and Hdac3 mRNA levels, but not Hdac1 levels in the hippocampus (Figure 3A ). This is consistent with the observation that HDAC2, but not HDAC1, is more important in binding to promoters of activity-related genes (Guan et al., 2009) . These results suggest that exercise may modulate Bdnf gene expression in the hippocampus by inducing changes to the epigenetic landscape of its promoter. To test this possibility, we performed chromatin immunoprecipitation experiments and showed that the binding of both HDAC2 and HDAC3 to the Bdnf promoter in hippocampi of exercise animals was decreased, in comparison to sedentary animals (Figure 3B and data not shown). Even though some variation was detected in the basal binding of HDAC3 to the Bdnf pI promoter, the overall data is consistent with exercise inducing a significant decrease in HDAC3 binding to the Bdnf promoter. As a result, these results focused our search for endogenous metabolites that are produced in response to physical activity and that may also act as an epigenetic modulator.
Exercise induces DBHB levels in the hippocampus Exercise is accompanied with increases in energy requirements leading to mitochondrial biogenesis, as well as higher metabolism and oxygen consumption. For example, exercise is often associated with increases in ketone body production. The ketone body D-b-hydroxybutyrate (DBHB) is a major energy metabolite that is increased in the liver after prolonged exercise. DBHB levels are frequently increased after caloric restriction, fasting and ketogenic diets and DBHB is believed to serve as a signaling molecule in response to metabolic changes (Newman and Verdin, 2014) . DBHB is synthesized from acetyl-CoA generated from the b-oxidation of fatty acids in the liver. As DBHB production is sensitive to environmental factors, nutritional states and energy levels, it has been hypothesized that ketone bodies may serve as an intermediary to regulate gene expression and chromatin structure. Interestingly, DBHB is transported in the blood stream to the brain where it can serve as an energy source. In addition, DBHB also has been shown to act as a class I HDAC inhibitor in non-neuronal tissues (Shimazu et al., 2013) . To assess the effects of voluntary running, we measured DBHB levels in the hippocampus following exercise. We found that an increase in DBHB levels occurred in mice after exercise, compared to mice not subjected to voluntary running (Figure 4A ).
DBHB induces BDNF expression To evaluate if DBHB had an effect upon Bdnf expression, we treated cortical neurons, which represent an abundant source of activity-dependent BDNF. Overnight treatment with DBHB significantly induced the coding and pI-driven Bdnf transcripts (Figure 4B ), consistent with the effects of exercise. Treatment of hippocampal slices with DBHB also gave similar results (Figure 4C ). To test the ability of DBHB to induce Bdnf in an in vivo setting, we administered exogenous DBHB intraventricularly in mice. Gene expression analysis indicated an increase specifically in Bdnf promoter I activity in the hippocampi of mice receiving the intraventricular injection of DBHB as compared to saline injections (Figure 4D ).
DBHB induces BDNF expression through HDAC2/HDAC3 inhibition and Histone H3 acetylation One possible explanation for the effects of exercise is that it induces DBHB accumulation in the hippocampus, which in turn inhibits class I HDACs such as HDAC2 and HDAC3 and leads to induction of Bdnf expression. To determine whether DBHB induction of Bdnf represents an HDAC-dependent mechanism, we tested whether DBHB treatment affected HDAC2 and HDAC3 occupancy at the Bdnf promoters. We find that treatment of primary neurons with DBHB led to decreased HDAC2 and HDAC3 binding to the Bdnf promoters (Figure 5A ). This was consistent with an increase in levels of acetylated histone H3 after DBHB treatment (Figure 5B ). Because DBHB is a potent inhibitor of HDAC3 (Shimazu et al., 2013) and HDAC2 has been previously shown to repress BDNF gene expression (Guan et al., 2009) , we focused our attention on HDAC3. Inactivation of HDAC3 either by treating cortical neurons with a HDAC3 selective HDAC inhibitor BRD3308 (Barton et al., 2014 ) Wagner et al., 2016 (Figure 5C) or through knockdown using shRNA targeting HDAC3 (Figure 5D, E, & F,E ) both led to increases in Bdnf expression. Taken together, these results suggest that inhibition of HDAC2 and HDAC3 in the hippocampus induces Bdnf expression.
DBHB links exercise induced metabolic changes in the liver to changes in gene expression in the hippocampus While brain cells catabolize glucose for energy under normal physiological conditions, ketone bodies are utilized when blood glucose levels decrease, as observed during exercise, fasting or caloric restriction. In order to mimic the increases in DBHB in the brain observed after exercise, we injected mice with 2-deoxy-d-glucose (2-DG), a structural analog of glucose that inhibits glycolysis and increases the brain's capacity to utilize ketone bodies as fuel. 2-DG is transported by glucose transporters into the cell where it binds to, but cannot be phosphorylated by hexokinase, resulting in the inhibition of the first step of glycolysis (Ralser et al., 2008) . This inhibition leads to the activation of a compensatory mechanism resulting in the production of ketone bodies by the liver. By activating this alternative energy pathway, 2-DG treatment induces DBHB levels in the brain. Interestingly, we found that 2-DG injection, like exercise, also induces both DBHB levels and Bdnf expression in the hippocampus to a similar extent (Figure 6A and B ). Interestingly, co treatments of a DBHB transporter inhibitor (AR-C155858) with 2-DG attenuated the 2-DG-induced DBHB levels and Bdnf expression. Hence, these findings are consistent with a model in which exercise induces DBHB . For the coding promoter, the n number for controls and BRD3308 treatments are 6 and 3 respectively. For the pI promoter, the n number for controls and BRD3308 treatments are 5 and 4 respectively. Each replicate consisted of primary neurons obtained from different cultures and treated with fresh dilutions of the compounds. Significance was measured by unpaired t-test p<0.01. (D) Hdac3 knockdown is verified by real-time RTPCR. N = 5 represents independent times knockdown was achieved in different primary culture using nucleofection. Significance was measured by unpaired t-test p<0.01. (E): HDAC3 knockdown is verified by western blotting. (F): HDAC3 knockdown significantly induces Bdnf coding gene expression as verified by real-time RTPCR. N = 5 and significance was measured by unpaired t-test *p<0.05. The Hdac3 shRNA sequence 2 that significantly reduced HDAC3 protein levels induced Bdnf expression. DOI: 10.7554/eLife.15092.007 accumulation in the hippocampus (Figure 6A and B ). DBHB stimulates histone acetylation at the Bdnf promoters through reduced HDAC2 and HDAC3 occupancy. This results in an increase in Bdnf gene transcription which reflects an epigenetic mechanism after exercise.
DBHB induces neurotransmitter release in the hippocampus To investigate the consequences of an increase of DBHB in the hippocampus, we conducted electrophysiological measurements in hippocampal slices to evaluate DBHB's effects on synaptic transmission. We incubated hippocampal slices with DBHB (0.8 mM) and then carried out field recording of post-synaptic potentials (fEPSPs) in CA1 evoked by stimulating the Schaffer collateral fibers (Yano et al., 2006) . Paired pulsed facilitation (PPF) was measured as the ratio of fEPSP slopes in response to two stimuli delivered to the Schaffer collateral inputs. PPF was followed after interstimulus intervals of 20-40 milliseconds. Incubation of hippocampal slices with DBHB (0.8 mM, 3 hrs) increased the fEPSP slope which was blocked by K252a (200 nM). Consistent with these results, DBHB decreased paired pulse ratio in a K252a-dependent manner suggesting a pre-synaptic modulation mediated by TrKB signaling (Figure 7A and B ). The actions of BDNF upon synaptic transmission are known to result in an increase in the frequency of synaptic currents, indicating a presynaptic role of BDNF (Park and Poo, 2013) . In fact, BDNF rapidly increases spontaneous neurotransmitter release in hippocampal neurons (Jovanovic et al., 2000; Li et al., 1998b) , which require TrkB receptors localized at pre-synaptic sites (Li et al., 1998) . These measurements indicated that DBHB treatment enhanced glutamatergic transmission at CA3-CA1 synapses (Figure 7 ) and that this DBHBinduced effect was blocked by K252a, a frequently used inhibitor of TrkB receptors in synaptic transmission experiments (Chen et al., 2015; Li et al., 1998b; Takei et al., 1998) . K-252a at 200 mM concentration specifically inhibits Trk receptors and not other tyrosine kinase receptors (Berg et al., 1992) . Hence the presynaptic enhancement by DBHB is dependent upon the BDNF TrkB receptors. These findings are consistent with an increase of BDNF by DBHB in the hippocampus and furthermore indicate there are additional physiological outcomes mediated by DBHB that involve an increase in neurotransmitter release.
Discussion These results provide a link between running exercise, the ketone body DBHB and Bdnf gene expression. Previous work with DBHB showed it was an effective neuroprotective agent in Huntington's disease (Lim et al., 2011 ) and Parkinson's disease (Kashiwaya et al., 2000; Tieu et al., 2003) , affecting striatal and dopaminergic neurons, respectively. It is highly conceivable that DBHB might act to increase the levels of BDNF, which can rescue neurons that are vulnerable in Huntington's and Parkinson's disorders (Autry and Monteggia, 2012) . Indeed, treatment of Alzheimer's disease 3xTgAD mice with 2-DG to induce ketone bodies delayed the progression of bioenergetic deficits in the brain and the associated b-amyloid burden. Similar to our experiments, 2-DG was capable of inducing BDNF in 3xTgAD mice (Yao et al., 2011) . Brain is a major source of the secretion of BDNF after exercise (Rasmussen et al., 2009) . Moreover, changes in the levels of BNDF have been suggested in the pathophysiology of schizophrenia, addiction and eating disorders (Autry and Monteggia, 2012) . A number of studies have shown that exercise can also improve depressive-like behavior through increased levels of hippocampal BDNF, which can enhance plasticity and synaptogenesis and reduce neurodegeneration (Aguiar et al., 2011; Cotman et al., 2007; Duman and Monteggia, 2006; Lu et al., 2013; Marais et al., 2009; Martinowich et al., 2007; Russo-Neustadt et al., 2000) . Given that histone acetylation in the hippocampus and cortex is associated with effects on Ketone bodies are widely distributed from the liver to the heart, muscle and the brain after fasting, dieting and intense exercise. When glucose levels are reduced, ketone bodies, produced in the liver from fatty acids in the form of DBHB and acetoacetate, serve as an energy source. In the brain, the levels of ketone bodies can reach very high levels (1-5 mM) (Mitchell et al., 1995; Robinson and Williamson, 1980) . A number of studies have demonstrated neuroprotective effects of ketone bodies in neurodegenerative and neuronal activity, such as under epileptic conditions (Maalouf et al., 2009) . It is probable there are multiple mechanisms that are involved in the effects of exercise in increasing trophic factor expression. For example, it has been reported that exercise induces Bdnf expression through the induction of hippocampal expression of Fndc5 (Wrann et al., 2013) , a PGC-1a and ERRa -dependent myokine. Once BDNF protein levels increase, TrkB signaling in turn inhibits Fndc5 expression in a negative feedback mechanism. We found prolonged exercise decreased Fndc5 expression levels consistent with elevated BDNF protein in the hippocampus (Wrann et al., 2013) . However, DBHB did not induce Pgc-1a, Fndc5, or Erra mRNA in primary neurons (data not shown), suggesting that there are alternative ways of affecting BDNF levels. The complexity of the BDNF gene and its alternative promoters and splicing events (Haberland et al., 2009) indicates that several regulatory mechanisms must exist to explain its many functions in synaptic transmission and neuronal survival (Park and Poo, 2013) . Interestingly, exercise induces Bdnf expression in the hippocampus and not in all the other regions of the brain. How this specificity is achieved is not clear. One possible explanation suggests that exercise may regulate the levels or functions of ketone body transporters in different regions of the brain. Indeed, acute exercise induces Mct2 transporter expression in the hippocampus immediately after and up to 10 hr post-exercise. This exerciseinduced upregulation of Mct2 was associated with increases in BDNF and TrkB levels (Takimoto and Hamada, 2014) . Our finding of an endogenous metabolite, DBHB, which upregulates Bdnf transcription in the hippocampus, provides one explanation linking the effects of exercise and peripheral metabolism to changes in epigenetic control and gene expression in the brain. There are multiple links between metabolic changes resulting from exercise and epigenetic modulation. For example, it has been reported that the blood levels of some Kreb's cycle intermediates such as a-ketoglutarate are increased after exercise (Leibowitz et al., 2012) . Interestingly, a-ketoglutarate is an essential cosubstrate for jumanji histone demethylases as well as for the tet proteins (DNA demethylases). Another molecule that may play a critical role in linking metabolic changes induced by exercise to epigenetic modulation is acetyl CoA which serves as the source of the acetyl group for all acetylation reactions catalyzed in the body including those catalyzed by histone acetyltransferases. Finally, coenzymes such as NAD+, or FAD produced during metabolic reactions are also required by sirtuins and the histone demethylase LSD1, respectively. In this paper, we provide evidence that an endogenous molecule, DBHB, that crosses the blood brain barrier, is increased by physical exercise to enhance the expression of a fundamental trophic factor in the brain and in turn affect synaptic transmission (Figure 8 ). Further studies aiming at identifying molecules that can also serve the dual purpose of an energy fuel and epigenetic modulator will help us accumulate additional members of the "exercise pill". The identification of these molecules is of great interest as many people afflicted with depression or with neurodegenerative diseases are likely to benefit from the ability of exercise to stimulate BDNF through small metabolites, such as DBHB. The involvement of ketone bodies in many other syndromes, such as glucose utilization, diabetes and epilepsy, suggests they represent vital molecules with broad metabolic effects upon chromatin and gene expression. B ) Average paired pulse ratio in control (6 slices/6 mice), DBHB (6 slices/6 mice), K252a (6 slices/3 mice), and K252a+DBHB (6 slices/3 mice) groups. Two-way repeated measures ANOVA showed significant difference between groups (F 3,20 = 4.03, p = 0.022). DOI: 10.7554/eLife.15092.009
Materials and methods
Exercise paradigm Male mice were individually housed with food and water ad libitum, lights on: 6 AM and lights off 6 PM. They were divided into 2 groups (Control and Running, n = 10 each); Control mice and running animals were housed with free access to a running wheel. Running distance and time were monitored (Coulborn Instruments). Animals were sacrificed after 30 days and hippocampus and cortex were collected and immediately frozen on dry ice. Animal care and use was in accordance with the guidelines set by the National Institutes of Health and the New York State Department of Health
Cell culture Immature primary cortical neurons were obtained from C57BL/6 mice [embryonic day 17 (E17)] as previously described (Ratan et al., 1994) . Mature cortical neurons were maintained in Neurobasal media (Invitrogen) supplemented with MACS NeuroBrew-21 (Miltenyi Biotec), and Glutamax (Invitrogen).
Cell treatment Primary neurons wereisolated as described above and 0.5 million cells were plated in six well plates. On Day 6, cells were treated with different concentrations of DBHB overnight or HDAC inhibitors for 5 hr. DBHB was prepared as 1 M stock in PBS and used at a final concentration of 5 mM. suberoylanilide hydroxamic acid (SAHA), Acetyldinaline (CI-994), Entinostat (MS-275), BRD3308 and BRD4097 were prepared as 10 mM stocks in DMSO and used at a final concentration of 10 mM. Trichostatin A (TSA) was prepared as a 670 mM stock and used at a final concentration of 0.67 mM.
RNA extraction and Real Time PCR Total RNA was prepared from primary cortical neurons or hippocampi usingthe NucleoSpin RNA II Kit (Clontech) according to the manufacturer's protocol. Real-time PCRs wereperformed using standard PCR protocol. Details of the Primers used are provided below: Primer sequence (5'-3'):
DBHB measurement DBHB levels were measured using a DBHB Assay kit (MAK041, Sigma) according to manufacturer's protocol.
Immunoblot analysis To determine HDAC3, acetyl HISTONE H3, and HISTONE H3 protein levels, total cell proteins were prepared by lysing cells in RIPA-B (1% Triton X-100, 1% SDS, 50 mM Tris-Cl, pH 7.4, 500 mM NaCl and 1 mM EDTA) in the presence of protease inhibitors (Sigma), the proteasome inhibitor MG-132 (Sigma) and phosphatase inhibitors (Sigma) followed by benzonase nuclease (Sigma) digestion for 15 min. Samples were boiled in Laemmlibuffer and electrophoresed under reducing conditions on NuPAGE Novex 4-12% for Bis-Tris Gel polyacrylamidegels (Invitrogen). Proteins were transferred to a nitrocellulose membrane(Bio-Rad) by electroblotting. Nonspecific binding was inhibitedby incubation in Odyssey blocking buffer (LI-COR Biosciences). Antibodies against HISTONE H3 (Cell signaling) and acetylated HISTONE H3 (Millipore), HDAC3 (ab16047, Abcam) and b-ACTIN (AC-74; Sigma-Aldrich) were diluted 1:1000, 1:1000, 1:1000; and 1:10,000, respectively, in odyssey blocking buffer and the membranes were incubated overnight at 4 ̊C. Fluorophore-conjugated Odyssey IRDye-680 or IRDye-800 secondary antibody (LI-COR Biosciences) was used at 1:10,000 dilution followed by incubation for 1 hr at room temperature. Finally, proteins were detected using an Odyssey infrared imaging system (LI-COR Biosciences). To determine BDNF protein levels, total hippocampal proteins were prepared by homogenizing tissues in RIPA-B in the presence of protease inhibitors, the proteasome inhibitor MG-132 and phosphatase inhibitors followed by benzonase nuclease digestion for 15 min. Samples (100mg) were boiled in Laemmlibuffer and electrophoresed under reducing conditions on NuPAGEÒ Novex 12% Bis-Tris Gel polyacrylamidegels. Proteins were transferred to a nitrocellulose membrane(Bio-Rad) by electroblotting. The membranes were washed twice with 1X PBS followed by 30 min incubation with 2.5% glutaraldehyde/PBS. The membranes were washed twice again with PBS and then with TBS-T. Nonspecific binding was inhibitedby incubation in 5% milk/TBS-T followed by incubation with the BDNF antibody (N-20, Santa Cruz) for 2 hr at room temperature. A peroxidase conjugated goat anti-rabbit or anti mouse was used for BDNF or actin detection, respectively. The membranes were detected by chemiluminescence.
Hippocampal slices
Intraventricular delivery of DBHB All surgeries were performed in accordance to IACUC rules. Mice were anesthetized under isoflurane and placed on a stereotaxic frame (David Kopf instruments, CA). The body temperature of the animals was maintained at 37 ̊C using a homeothermic blanket. DBHB (2 mM and 5 mM; 5 ml) was delivered by a Hamilton syringe at a flow rate of 0.5 ml/min using a nanomite syringe pump (Harvard apparatus, MA). The stereotaxic coordinates relative to bregma were as follows: AP, -0.46 mm; L, -1.20 mm; DV, 2.20 mm. (Paxinos and Watson, 1998) . In sham control animals, 5 ml of saline (vehicle) were infused. Proper post-operative care was taken until the animals recovered completely. Mice were sacrificed after 6 hr following the injection and the different brain regions were dissected.
Hdac3 short hairpin RNA knockdown Hdac3 (NM_010411) short hairpin RNA (shRNA) clone (TRCN0000039391, 5' CCGGGTGTTGAATATGTCAAGAGTTC TCGAGAACTCTTGACATATTCAACACTTTTTG 3 0 ; Sigma) and Non-Target shRNA Control Vector (Sigma) were introduced into immature primary cortical neurons (E17) using the Amaxa mouse Neuron Nucleofector kit as directed by the manufacturer (Lonza). On Day 6, HDAC3 knockdown was confirmed by Real-time RTPCR and whole-cell lysate Western blots.
Chromatin immunoprecipitation The Ez-Magna ChIP assay kit was used as directed by the manufacturer (Millipore). Briefly, primary cortical cells were crosslinked with 1% formaldehyde at 37 ̊C for 7 min. Cells were then sonicated using the Bioruptor (Diagenode) and immunoprecipitated with primary antibodies (10 mg). The crosslinking was reversed, and the DNA was isolated on the columns provided by the kit. Shearing size was determined to be between 150 and 1000 bp. Real-time PCR was conducted with primers targeted to the BDNF pI promoter (TGATCATCACTCACGACCACG and CAGCCTCTCTGAGCCAG TTACG) and SYBR Green PCR Master mix (Applied Biosystems). Each experiment was conducted at least three times by crosslinking cells from different primary cortical neuron preparations.
2-deoxy-d-glucose injections mice received intraperitoneal injections of 10mg/Kg of 2-DG or saline. They were left to recover for 6 hrs after which the animals were sacrificed and the tissue was harvested. Electrophysiology 2-4 months old C57BL6 male mice were anesthetized by pentobarbital anesthesia. After decapitation, hippocampi were isolated in ice-cold artificial cerebrospinal fluid (ACSF) containing the following (in mM), NaCl (118), KCl (4.5), glucose (10), NaH 2 PO 4 (1), CaCl 2 (2) and MgCl 2 (2) (aerated by 95%O2/5% CO2, pH adjusted to 7.4 with NaHCO 3 ). Hippocampal slices (300 mm) were prepared on a vibratome (Campden Instruments) and maintained at room temperature for 1 hr in a brain slice keeper. Brain slices were incubated in vehicle (0.00007% DMSO), K252a (200 nM), DBHB (0.8 mM) or combination of K252a and DBHB for 3 hrs at room temperature before recording CA1 field excitatory post-synaptic potentials (fEPSPs) in a recording chamber perfused with ACSF at 32 ̊C. fEPSPs were evoked by stimulation of the Schaffer collateral fibers using a concentric bipolar electrode (FHC). Input-output data were generated by plotting fEPSP slope in response to 100, 200, 300, 400 and 500 mA stimulation. Paired pulse ratio was measured as ratio of second fEPSP slope to first EPSP slope at 20, 40, 80, 120 and 160 ms inter-stimulus interval. A response approximately 35% of the maximum evoked response was used for studying paired pulse ratio. Data were acquired using pCLAMP 10 program and MultiClamp 700B amplifier (Molecular Devices). Data analysis was carried out using Clampfit program.
Statistical analysis unpaired t-test, 1way or 2way ANOVA followed by the Dunnett or Bonferroni post tests respectively were used to measure statistical significance. p<0.05 was considered to be statistically significant. Figure 1 . 1 Figure 1. The experimental design and time course of the exercise regime is shown. Exercise induces changes in brain BDNF levels in a voluntary exercise protocol (16). (A) Experimental design for the Voluntary exercise model.Figure 1 continued on next page
Figure 1. The experimental design and time course of the exercise regime is shown. Exercise induces changes in brain BDNF levels in a voluntary exercise protocol (16). (A) Experimental design for the Voluntary exercise model.Figure 1 continued on next page
Figure 2 . 2 Figure 2. HDAC inhibitors induce Bdnf expression. (A) Broad spectrum HDAC inhibitors such as TSA (0.67 mM) and SAHA (5 mM) induce coding and pI Bdnf expression levels as measured by real-time RTPCR. For the coding promoter, the n number for controls, TSA and SAHA treatments are 6, 5 and 4 respectively. For the pI promoter, the n number for controls, TSA and SAHA treatments are 5, 5 and 4 respectively. Each replicate consisted of primary neurons obtained from different set cultures and treated with fresh dilutions of the compounds. Significance was measured by 1way anova p< 0.01.and *p<0.001. (B) Treatment with class I HDAC inhibitors such as CI-994 (10 mM) and MS-275 (10 mM) induce coding and pI Bdnf levels, whereas the negative control analog for CI-994, BRD4097 does not as measured by real-time RTPCR. For the coding promoter, the n number for Figure 2 continued on next page
Figure 2 2 Figure2continued controls, CI-994, BRD4097 and MS-275 treatments are 6, 6, 5 and 5 respectively. For the pI promoter, the n number for controls, CI-994, BRD4097 and MS-275 are 5, 6, 5 and 5 respectively. Each replicate consisted of primary neurons obtained from different cultures and treated with fresh dilutions of the compounds. Significance was measured by 1way anova p<0.01.and *p<0.001. DOI: 10.7554/eLife.15092.004
Figure 4 . 4 Figure 4. Exercise increases DBHB levels in the hippocampus. DBHB in turn can induce Bdnf expression in vitro in neurons and in vivo in the hippocampus (A) Exercise induced DBHB levels in the hippocampus. The number for controls and exercise hippocampi is 10 and 11 respectively. Statistical significance was analyzed by the unpaired t-test *p<0.05. DBHB amounts are expressed as millimole per gram of total hippocampal protein (B) Overnight treatment of DIV6 primary cortical neurons with 5 mM of DBHB significantly induces coding and pI Bdnf expression levels as measured by real-time RTPCR. For both promoters, the n number for controls and DBHB are 4 and 4 respectively. Each replicate consisted of primary neurons obtained from different cultures and treated with fresh dilutions of the compounds. Significance was measured by unpaired t-test *p<0.05 and p<0.01. (C) Three hour treatment of hippocampal slices with DBHB (0.8 mM) induces coding and pI Bdnf levels as measured by real-time RTPCR. For both promoters, the n number for controls and DBHB are 5 and 5 respectively. Each replicate consisted of slices obtained from different animals and treated with fresh dilutions of the compounds. Significance was measured by unpaired t-test *p<0.05 and p<0.01. (D) Intraventricular delivery of DBHB (2 mM) significantly induces coding and pI Bdnf levels as measured by real-time RTPCR. The n number for controls and DBHB are 3 and 5 respectively. Each replicate consisted of hippocampi from different animals that were subjected to the surgical procedure. Significance was measured by unpaired t-test *p<0.05. DOI: 10.7554/eLife.15092.006
Figure 5 5 Figure 5 continued chromatin immunoprecipitations for each HDAC for each treatment were 3 (control), 3 (DBHB 2 mM) and 2 (DBHB 5 mM). Statistical significance was measured by 1way anova p<0.001. (B) HISTONE H3 acetylation is increased in neurons upon treatment with different doses of DBHB. Representative blot shown in the figure. (C) The HDAC3 selective inhibitor BRD3308 significantly induces Bdnf pI expression as measured by real-time RTPCR.. For the coding promoter, the n number for controls and BRD3308 treatments are 6 and 3 respectively. For the pI promoter, the n number for controls and BRD3308 treatments are 5 and 4 respectively. Each replicate consisted of primary neurons obtained from different cultures and treated with fresh dilutions of the compounds. Significance was measured by unpaired t-test p<0.01. (D) Hdac3 knockdown is verified by real-time RTPCR. N = 5 represents independent times knockdown was achieved in different primary culture using nucleofection. Significance was measured by unpaired t-test p<0.01. (E): HDAC3 knockdown is verified by western blotting. (F): HDAC3 knockdown significantly induces Bdnf coding gene expression as verified by real-time RTPCR. N = 5 and significance was measured by unpaired t-test *p<0.05. The Hdac3 shRNA sequence 2 that significantly reduced HDAC3 protein levels induced Bdnf expression. DOI: 10.7554/eLife.15092.007
Figure 6 . 6 Figure 6. DBHB can serve as an exercise factor linking metabolic changes in response to exercise to changes in gene expression in the brain. (A) 2-DG treatment known to induce ketone bodies in the brain induces Bdnf pI Figure 6 continued on next page
Figure 6 6 Figure 6 continued expression in the hippocampus as measured by real-time RTPCR. This effect was blocked by a DBHB transporter inhibitor. N = 10 (Ctrl), N = 9 (2-DG injected) and N = 5 (2-DG and AR-C155858). Significance was measured by unpaired t-test *p<0.05. (B) 2-DG treatment induced DBHB levels in the hippocampus and this effect is blocked by a DBHB transporter inhibitor. DOI: 10.7554/eLife.15092.008
Figure 7 7 Figure7continued significant difference between groups (F 3,20 = 10.13, p<0.001). Upper panel shows examples fEPSP traces. (B) Average paired pulse ratio in control (6 slices/6 mice), DBHB (6 slices/6 mice), K252a (6 slices/3 mice), and K252a+DBHB (6 slices/3 mice) groups. Two-way repeated measures ANOVA showed significant difference between groups (F 3,20 = 4.03, p = 0.022). DOI: 10.7554/eLife.15092.009
Figure 8 . 8 Figure 8. A proposed model by which exercise induces Bdnf expression in the hippocampus. Exercise induces DBHB synthesis in the liver. DBHB is transported through the circulation to peripheral organ including the brain. In the hippocampus, DBHB induces Bdnf expression through a mechanism involving HDAC inhibition. This induction in turn mediates exercise's positive effects on memory, cognition and synaptic transmission. DOI: 10.7554/eLife.15092.010
Animal care and use was in accordance with the guidelines set by the National Institutes of Health and the New York State Department of Health. 5-week old male mice were anesthetized and decapitated. The brain was dissected and placed in 4 ̊C cutting buffer (126 mM sucrose, 5mM KCl, 2 CaCl 2 , 2 mM MgSO 4 , 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , and 10 mM D-glucose, pH 7.4). The hippocampus was dissected and submerged in ice-cold cutting buffer and cut horizontally into 300-mm sections, which were immediately placed in recovery solution {50% cutting solution/ 50% artificial cerebrospinal fluid (ACSF)} and oxygenated (95% CO 2 -5% O 2 ) for 20 min. The slices were then transferred in to ACSF oxygenated chambers and treated with DBHB (0.8 mM concentration) for 3 hr after which RNA was extracted for Real-Time RT PCR analysis.
Sleiman et al. eLife 2016;5:e15092. DOI: 10.7554/eLife.15092
|
10.1074/jbc.m407886200
|
cc-by
| null | null |
openalex
|
The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for ␥H2AX in DNA repair; however, ␥H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on ␥H2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish ␥H2AX functions. We found that integration promotes transient formation of ␥H2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of ␥H2AX in chromatin repair.The evolutionarily conserved histone H2AX comprises approximately 2-25% of the histone H2A pool in mammalian cells and is incorporated randomly into nucleosomes (1) . The ex-tended C-terminal tail of H2AX contains a serine (Ser-139) embedded in an invariant SQE motif that is a target for phosphorylation by the phosphatidylinositol 3-kinase-related DNA-PK, ataxia-telangiectasia-mutated (ATM), and ATM and Rad3related (ATR) protein kinases (2) (3) (4) . This H2AX serine residue is massively and rapidly phosphorylated at sites of double strand breaks (DSBs) 1 and stalled replication forks (3, 5, 6) , forming microscopically visible foci on staining with a specific antibody. This phosphorylation seems to play an important role in processing or repair of DSBs (7, 8) . H2AX phosphorylation has also been observed at sites of V(D)J recombination (9) , meiotic strand breaks, and other physiologically programmed reactions in which DSBs are formed (10 -12) . Early events in retroviral replication include entry of the viral capsid with the accompanying enzymes reverse transcriptase and integrase (IN) followed by synthesis of a DNA copy of the viral RNA genome to form a preintegration complex. This complex then enters the nucleus, and integration is first detected at approximately 3-4 h postinfection (13) . Retroviral integration is catalyzed by integrase acting on specific sequences at the ends of the viral DNA and via a concerted cleavage-ligation reaction that is mechanistically similar to that catalyzed by RAG proteins during V(D)J recombination (14 -16) (Fig. 1A ). As a consequence of integrase-mediated joining, the host cell DNA suffers a DSB, but the ends are held together by single strand links to viral DNA (Fig. 1B ). Postintegration repair of this intermediate (Fig. 1B ) is essential for the maintenance of host DNA integrity as well as the stable association of retroviral DNA with host chromosomes. Numerous lines of evidence (17) (18) (19) (20) indicate that retroviral DNA elicits a DNA damage response and that the integration intermediate is repaired primarily via components of the non-homologous end-joining (NHEJ) pathway. In this study, we asked whether H2AX is phosphorylated at sites of retroviral DNA integration and whether this response is essential for repair of this complex lesion as determined by survival of stably transduced cells.
EXPERIMENTAL PROCEDURES Cells and Viruses-MO59K cells (purchased from American Type Culture Collection) were maintained as described previously (18, 19) , and mouse embryonic fibroblasts (MEFs) were maintained as described previously (17) . The ASV-based vectors IN ϩ and IN Ϫ and the HIV-1based vector were described previously (17, 19) . Immunofluorescence and Quantification of Foci-Cells were plated and infected the following day at multiplicity of infection (m.o.i.) 10. The cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde at the indicated times postinfection. After permeabilization with 0.2% Triton X-100, samples were blocked overnight with 3% bovine serum albumin at 4 C. The slides were incubated with a mouse monoclonal antibody against ␥H2AX (Upstate) and then with Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes) as secondary antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole prior to mounting the slides with SlowFade antifade reagent (Molecular Probes). Processed cells were examined for ␥H2AX foci by monitoring fluorescence of the Alexa Fluor dye using an UltraView confocal imaging system (PerkinElmer Life Sciences) in which the confocal scanning head was mounted on a Nikon TE-200E microscope. Optical sections along the z axis of the nuclei were captured at 0.1-m intervals, and the final images were obtained by projection of the individual sections. For each time point, images of at least 100 cells were captured and used for quantitative analysis of ␥H2AX foci. To prevent bias in the selection of cells that displayed foci, nuclei were randomly selected for 4,6-diamidino-2-phenylindole staining and then monitored for focus formation. The ␥H2AX foci were counted in each cell using Image-Pro Plus (Media Cybernetics). A setting that excluded relatively weak foci and background speckles was used as a standard for foci quantitation in all the cells selected for analysis. A simple stochastic model for viral integration and formation of ␥H2AX foci was designed to relate the number of foci counted in cells to the time of observation. Virus is modeled as being integrated at 4 h, on average, after the start of the experiment. The exact time of integration and start of H2AX phosphorylation are taken to be normally distributed, with a mean of 4 h and a standard deviation of 1.1 h. H2AX phosphorylation is modeled to continue for an exponentially distributed random time, with a mean of 2.6 h. The number of viruses integrating in a cell is taken to be Poisson distributed. The best fit to experimental data is achieved with a mean m.o.i. of 8.5 viruses/cell. Chromatin Immunoprecipitation-Chromatin immunoprecipitation (ChIP) assays were performed as described by Boyd and Farnham (21) . In our experiments, 10 6 HeLa cells were infected with amphotropic ASV vectors IN ϩ or IN Ϫ (17). At defined times after infection, formaldehyde (1% final) was added, and the cultures were incubated at room temperature for 30 min to cross-link viral DNA and interacting proteins. The cross-linking reaction was quenched with glycine (0.125 M final concentration). Plates were washed with cold 1ϫ phosphate-buffered saline, and then cells were scraped into 1ϫ phosphate-buffered saline that contained protease inhibitors and washed and lysed by addition of 0.5% Nonidet P-40, 5 mM PIPES, pH 8.0, 85 mM KCl, and protease inhibitors. The intact nuclei were isolated by centrifugation at 5000 rpm at 4 C. Nuclei were then resuspended in a lysis buffer (1% SDS, 50 mM Tris-Cl, pH 8.1, 10 mM EDTA, protease inhibitors). Chromatin was sonicated to fragments containing DNA of an average length of approximately 600 bp. Samples were subjected to centrifugation to remove debris and were precleared by shaking for 1 h with salmon sperm DNA/protein Aagarose (Upstate). After removal of the salmon sperm DNA/protein A-agarose, supernatants were diluted 10-fold with a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl, protease inhibitors), and chromatin fragments were immunoprecipitated overnight with antibodies to ASV integrase (rabbit polyclonal), ␥H2AX (mouse monoclonal) (Upstate), and phosphatidylinositol 3-kinase p110 (mouse monoclonal) (Santa Cruz Biotechnology) proteins. Protein-DNA-antibody complexes were isolated by the addition of salmon sperm DNA/protein A-agarose. After 1 h, complexes were collected by centrifugation and washed three times with a wash buffer (100 mM Tris, pH 8, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholic acid). Pellets were eluted with 50 mM NaHCO 3 , 1% SDS for 15 min at room temperature. Clarified samples were incubated with RNase and 5 M NaCl at 67 C for 4 -5 h to reverse cross-links and then precipitated overnight with ethanol. Following centrifugation, pellets were resuspended in proteinase K buffer and treated with proteinase K. After phenol/chloroform extraction, the DNA was precipitated with ethanol. Viral sequences in these fractions were detected by PCR using primers targeting the long terminal repeats. The sequence of the left primer was 5Ј-ACG TCC AGG GCC CGG AGC GAC-3Ј, and the right primer was 5Ј-CTT CAA TGC CCC CAA AAC CAA-3Ј. PCR products were resolved by electrophoresis on an agarose gel and subjected to Southern blotting with a radioactive probe against the ASV long terminal repeat (generated by using random primers and a PCR fragment made with the long terminal repeat probe primers 5Ј-GAT TGG TGG AAG TAA GGT GG-3Ј and 5Ј-CAA ATG GCG TTT ATT GTA TCG-3Ј). As a negative control, we used primers targeting the cellular p21 promoter sequences, left, 5Ј-TTT CCA CCT TTC ACC ATT CC-3Ј, and right, 5Ј-GGC AGA TCA CAT ACC CTG TT-3Ј, with a probe generated by using random primers. Transduction Assays-For infection with the ASV vector (22) , MEFs were plated at a density of 10 5 cells/60-mm dish. On the following day, cells were infected in the presence of 5 g/ml DEAE-dextran. At 8 days postinfection, EGFP-positive cells were counted by flow cytometry. For infection with the HIV-1 vector, MEFs were plated at a density of 5 ϫ 10 4 cells/well in a 24-well plate. The following day, cells were infected with an HIV-1 EGFP vector (23) . Transduced cells were counted by flow cytometry as with the ASV vector.
RESULTS To determine whether retroviral infection induces formation of ␥H2AX foci, we infected cells with an amphotropic ASV vector (17) and examined them by immunofluorescence with an antibody specific for ␥H2AX. In preliminary experiments (data not included), we observed the formation of ␥H2AX foci early after infection in DNA repair-proficient human (HeLa and MO59K) and mouse (3T3) cells. As a control, we infected cells with an integration-deficient (IN Ϫ ) vector (17) , and no increase in foci over background was detected. The data in Fig. 2 and Table I summarize results from subsequent experiments, which included computer-assisted quantitative analyses of ␥H2AX foci in MO59K cells infected at m.o.i. 10 infectious particles/cell. We again observed an increased number of foci in the infected culture (Fig. 2A ), which appeared to peak at 6 h postinfection. A comparison of the distribution of the number of foci/cell in uninfected cells with the number of foci/cell 6 h postinfection is shown in Fig. 2B . The bulk (ϳ75%) of the uninfected cells contained no or few foci/cell, whereas a small proportion (ϳ10%) displayed numerous foci, which we speculate may have been caused by replication stress; such cells were also observed in the infected culture. At 6 h postinfection, the infected culture had many fewer cells with no foci, and the percentage of cells with 5-10 foci was substantially higher than in the uninfected culture. As summarized in Table I , this value increased sharply by 4 h postinfection, when integration is expected to begin. The average number of virus-induced foci peaked at 5.1 foci/cell in the 6-h sample and declined again at 8 h postinfection. ␥H2AX foci are reported (6) to arise within minutes at sites of DNA damage and start to disappear after 30 min, with an ϳ2-h half-life as damage is repaired. Assuming similar kinetics, it is likely that some integration-induced foci were both formed and resolved within the 8-h interval monitored in this experiment. A computer simulation using such parameters produced data consistent with the numbers of virus-induced foci/cell in a culture infected at m.o.i. ϳ10, at the postinfection time points shown in Table I . To verify that H2AX is phosphorylated at sites of retroviral DNA integration, we immunoprecipitated chromatin from nuclear extracts with a ␥H2AX-specific antibody (ChIP assay) and screened for the presence of viral DNA using PCR. To test the feasibility of this approach, ChIP was first performed at 6 h postinfection of HeLa cells at m.o.i. 0.001, 0.01, or 0.1 infectious particles/cell (Fig. 3A , lanes 2-4). As expected, antibody specific for integrase protein (positive control) precipitated viral DNA in this extract and in amounts proportional to the m.o.i. An association of viral DNA with the ␥H2AX immunoprecipitate was also readily detected and, as with integrase, in proportion to the m.o.i. Approximately 10% of the viral DNA was coimmunoprecipitated with ␥H2AX in this test. Based on the calculated efficiency of the antibody used for these analyses (data not shown), the actual amount of nuclear viral DNA associated with ␥H2AX in this experiment is estimated to be ϳ37% of the total. No association of viral DNA was detected with the phosphatidylinositol 3-kinase antibody (negative control). In addition, none of the antibodies precipitated sequences corresponding to a region in the cellular p21 promoter (DNA negative control). In this experiment, viral DNA was quantified by Southern blot, but comparable results were obtained with real time PCR (not shown). Because results with m.o.i. 0.1 are clearly in the titratable range for ChIP analysis, this was the condition adopted in the two separate experiments summarized in Fig. 3, B and C . Fig. 3B shows results of ChIP analyses following infection with integration-competent (IN ϩ ) or integration-defective (IN Ϫ ) ASV vectors. A peak of association of ␥H2AX and viral DNA was observed in the IN ϩ infection at 6 h postinfection, and no association was detected after infection with the IN Ϫ vector. This result demonstrates that ␥H2AX is associated with nuclear viral DNA only after this DNA is integrated into chromatin. To examine more closely the kinetics of accumulation of ␥H2AX at integration sites, the amount of viral DNA captured by ChIP at 2-h intervals was determined up to 16 h postinfection (Fig. 3C ). Values for each time point were expressed as a percentage of the total viral DNA in the nuclear fraction that is associated with the ChIP at each time, corrected for immunoprecipitation efficiency of the ␥H2AX antibody. Consistent with our analysis of foci in infected MO59K cells (Table I ), these results showed that an association with ␥H2AX peaks at ϳ6 h, shortly after viral DNA is detected in the nuclear extract. We estimate that 60% of the nuclear viral DNA in this experiment is associated with ␥H2AX and therefore is integrated at this time point. From these results, we conclude that ␥H2AX is a valid marker for sites of retroviral DNA integration. We note that ChIP detects integrated viral DNA both before and after repair (Fig. 1 ). To examine the functional relevance of H2AX phosphorylation to repair, we performed the transduction assays described below. Because transduced genes are expressed efficiently only from stably integrated proviruses, retroviral transduction is a readout for successful postintegration repair. For example, we have shown that the transduction efficiency of NHEJ-defective DNA-PKcs-deficient murine cells is reduced by 80 -90% com-
TABLE I Quantitative assessment of ␥H2AX foci formation in response to retroviral infection in MO59K cells Cells were infected at an m.o.i. of 10, fixed with paraformaldehyde at the indicated times following infection, and immunostained with ␥H2AX antibody. The number of foci/cell as well as the percentage of cells displaying foci for an 8-h time course postinfection were determined as described under "Experimental Procedures." The number of virus-induced foci/cell was determined by subtracting the background foci in uninfected cells (time 0) from the various time points. pared with wild type cells or deficient cells into which DNA-PKcs-expressing DNA was reintroduced (17, 20) . As the DNA damage induced by integration cannot be repaired efficiently, most NHEJ-deficient cells are unlikely to survive infection and therefore cannot give rise to stable transductants. To find out whether H2AX phosphorylation is required for postintegration repair, we performed transduction experiments using MEF lines obtained from H2AX knock-out mice and derivatives (24, 25) , and the ASV-EGFP vector. Results in Table II show that there is no significant difference between the transduction efficiency with H2AXϩ/ϩ and H2AXϪ/Ϫ MEFs. Similar results were obtained with a VSV-G protein-pseudo-typed HIV-1 GFP vector (not shown). These data indicate that H2AX deficiency has little or no effect on postintegration repair. One possible explanation for this result is that H2AX function in these cells is redundant. We therefore examined transduction in H2AXϪ/Ϫ MEFs that had been complemented with transgenes that express wild type murine H2AX or proteins carrying either neutral non-modifiable substitutions (S136A/S139A) or negatively charged substitutions that mimic constitutive phosphorylation (S136E/S139E) in the C-terminal phosphatidylinositol 3-kinase-related protein kinase target sites of H2AX. No significant difference was observed between the efficiencies of transduction of these lines compared with H2AXϪ/Ϫ cells (Table I ). It appears, therefore, that H2AX phosphorylation is dispensable for postintegration repair.
DISCUSSION In this study, we show that retroviral infection induces the formation of histone ␥H2AX foci, and chromatin immunoprecipitation assays confirmed that H2AX phosphorylation occurs at sites of retroviral DNA integration. Therefore, these results are consistent with our previous findings (17) (18) (19) (20) that cells respond to retroviral DNA integration in a manner similar to DSBs. We also demonstrate that an H2AX deficiency and an inability to phosphorylate this histone have no detectable effect on the efficiency of retroviral transduction of cultured mouse cell lines. Because efficient expression of transduced genes
TABLE II Transduction efficiency of H2AX null cells and null cells complemented with H2AX genes carrying mutation of the C-terminal phosphorylation site of this histone Cells were infected with an amphotropic ASV vector carrying an EGFP reporter (23) . Virus titer ϳ1.4 ϫ 10 5 infectious particles/ml. wt, wild type. requires stable retroviral vector integration, we conclude that H2AX phosphorylation is largely dispensable for postintegration repair of chromatin damage. Although ␥H2AX is required for the accumulation of a subset of repair and signaling proteins into irradiation-induced foci (7, 8, 24, 26, 27) , the exact role of H2AX phosphorylation is not well understood. Some DNA damage-sensing and repair proteins have been shown to interact physically with ␥H2AX (27) (28) (29) . A functional role for ␥H2AX has been indicated because H2AX-deficient cells are hypersensitive to ionizing radiation, exhibit genomic instability, and also show an aberrant checkpoint response under certain conditions (7, 8, 30) . On the other hand, although ␥H2AX foci form at sites of V(D)J recombination, H2AX appears to be dispensable for this reaction when tested with extrachromosomal substrates in cultured cells (7, 8, 30) . The study of H2AX-deficient mice has provided further insight into H2AX function. H2AX mice are viable, but DNA repair seems to proceed less efficiently in such animals, which show modest sensitivity to ionizing radiation and impairment in immunoglobulin class-switch recombination (31) . In keeping with results from the cell-based assays cited above, these mice show no detectable abnormalities in V(D)J recombination (7, 8) . However, the genomic caretaker function of H2AX is more fully exposed when cell cycle checkpoints are compromised because of the absence of p53 (25, 32) . In a p53Ϫ/Ϫ background, even H2AXϩ/Ϫ heterozygotes show increased misrepair of DNA damage, leading to the development of immature T-and B-cell lymphomas and solid tumors. Some of the B-cell lymphomas harbor oncogenic translocations with hallmarks of aberrant V(D)J recombination. It appears therefore that, although H2AX is not required for V(D)J recombination, it can suppress misrepair of RAG-dependent DSBs. Based on these and other observations, two general, non-exclusive models have been proposed for ␥H2AX function. 1) The high concentration of repair proteins recruited to the vicinity of DSBs by or through some action of ␥H2AX may facilitate repair, especially at low (threshold) levels of damage (30); or 2) ␥H2AX interaction with such proteins might help to hold broken ends together, thereby minimizing the risk of misrepair (25, (32) (33) (34) . According to these models, differential dependency on H2AX is expected, with more dependence on ␥H2AX for the repair of free DSBs formed by ionizing radiation than for programmed recombination reactions (e.g. V(D)J), in which ends are held in proximity by recombination proteins (35) . H2AXϪ/Ϫ spermatocytes show severe defects in meiotic X-Y chromosome pairing (12) , and as such, it is tempting to speculate that ␥H2AX plays a bridging function during meiotic recombination. In summary, our studies have produced two important findings. First, they provide direct confirmation that cultured cells respond to retroviral DNA integration in the same way that they respond to DSBs produced by a variety of genotoxic agents or normal programmed events, namely, by massive phosphorylation of histone H2AX in the vicinity of the damage site. The second finding is that H2AX appears to be dispensable for postintegration repair. These observations lend independent support to a model in which the anchoring of broken DNA ends to facilitate their repair is a critical function of ␥H2AX. Because chromosome breaks are likely to be held together by the RAG1/2 complex during V(D)J recombination and are held covalently by viral DNA in retroviral integration (Fig. 4 ), such an anchoring function should not be essential in either reaction. FIG. 1 . 1 FIG. 1. Stable, heritable establishment of a retroviral provirus requires integrase-mediated DNA integration and postintegration repair of the integration intermediate by host proteins. A, the first two steps in this process are catalyzed by a multimer of integrase, (IN)n. B, postintegration repair is dependent on host cell functions.
FIG. 2 . 2 FIG. 2. Retroviral infection induces formation of ␥H2AX foci. A, confocal images show uninfected MO59K cells (left) and 6 h postinfection with an ASV vector at m.o.i. 10 (right). Nuclei were stained with 4,6-diamidino-2-phenylindole (blue). B, distribution of foci is shown in uninfected cells (open bars) and in cells 6 h after infection (filled bars).
FIG. 4 .FIG. 3 . 43 FIG.4. Broken chromosomal ends may be held together by several mechanisms to facilitate NHEJ. Proximity of ends produced by ionizing radiation (IR) or treatment with genotoxic agents would be enhanced by ␥H2AX-mediated anchoring via specific proteinprotein interactions. Ends produced during V(D)J recombination would also be held together by the RAG1/2 protein complex (dashed oval). Ends produced during retroviral integration are linked by single stand covalent bonds to viral DNA and, perhaps, by the viral IN complex as well.
The numbers in parentheses are from the computer simulation described under "Experimental Procedures." Time Percentage of cells No. of No. of virus postinfection with foci foci/cell induced foci/cell h 0 53 5.2 0.0 (0.00) a 2 51 5.5 0.3 (0.26) 4 92 8.6 3.4 (3.41) 6 88 10.3 b 5.1 (4.88) 8 84 8.2 3.0 (3.05) a b p Ͻ 10 Ϫ6 for this time point.
The abbreviations used are: DSB, double strand break; IN, integrase; ASV, avian sarcoma virus; HIV, human immunodeficiency virus; m.o.i., multiplicity of infection; ChIP, chromatin immunoprecipitation; PIPES, 1,4-piperazinediethanesulfonic acid; NHEJ, non-homologous end-joining; EGFP, enhanced green fluorescent protein; VSV, vesicular stomatitis virus; MEFs, mouse embryonic fibroblasts.
This paper is available on line at http://www.jbc.org
|
10.2471/blt.18.216119
|
cc-by
| null | null |
openalex
|
Home-based records have been used in both low-and high-income countries to improve maternal and child health. Traditionally, these were mostly stand-alone records that supported a single maternal and child health-related programme, such as the child vaccination card or growth chart. Recently, an increasing number of countries are using integrated home-based records to support all or part of maternal and child health-related programmes, as in the maternal and child health handbook. Policy-makers' expectations of home-based records are often unrealistic and important functions of the records remain underused, leading to loss of confidence in the process, and to wasted resources and opportunities for care. We need to examine the gaps between the functions of the records and the extent to which users of records (pregnant women, mothers, caregivers and health-care workers) are knowledgeable and skilful enough to make those expected functions happen. Three key functions, with increasing levels of complexity, may be planned in home-based records: (i) data recording and storage; (ii) behaviour change communication, and (iii) monitoring and referral. We define a function-capacity conceptual framework for home-based records showing how increasing number and complexity of functions in a home-based record requires greater capacity among its users. The type and functions of an optimal home-based record should be strategically selected in accordance not only with demands of the health system, but also the capacities of the record users.Introduction In September 2018, the World Health Organization (WHO) launched its Recommendations on home-based records for maternal, newborn and child health. 1 The guidelines reconfirmed the effectiveness of home-based records in increasing the use of maternal and child health services. This initiative by WHO provides the global health community with an opportunity to revisit what these records are and how they should be designed and implemented. A home-based record is a health document used to record the history of health services received by an individual. The record is kept in the household, in paper or electronic format, by the individual or his or her caregivers. The record is intended to be integrated into the routine health information system and to complement records maintained by health facilities. Home-based records have been used in over 163 countries, notably for the improvement of maternal and child health. 1 Traditionally, maternal and child health programmes have developed their own programme-specific home-based records and implemented these in parallel as an essential part of their vertical service-delivery systems. Examples include child vaccination cards for child immunization programmes, 2,3 growth charts for child nutrition programmes 4 and maternal health cards for reproductive and maternal health programmes. 5 In some countries, the child vaccination card and growth chart are integrated into a child-targeted home-based record, often called the child health card or handbook. 1 In many countries, the maternal health card continues to be a stand-alone record covering pregnancy, childbirth and the postpartum stages, independent from child records. We have observed that this difference in target groups has prompted policy debates on whether home-based records should be developed and distrib-uted per child or per mother. Moreover, even if the consensus is for full integration of all the maternal and child records into a single record, poor coordination across departments within a health ministry can make it difficult to enforce such integration due to the nature of vertical programmes. Most countries therefore prefer to implement a series of programmespecific, stand-alone home-based records for maternal and child health services. In efforts to address the millennium development goals, there has been an increasing interest in integrated records that cover the entire spectrum of pregnancy, childbirth, postpartum and newborn care, through to childhood and, sometimes, adolescence. As of 2016, at least 25 countries used fully integrated home-based records, often called the maternal and child health handbook. 6 Key advantages of full integration include greater assurance of a continuum of maternal and child health care and major cost savings in the operation of the records. 11 Fully integrated home-based records have been attracting more attention from health ministries 12, 13 and professional organizations 14, 15 as an effective tool for promoting a life-course approach to health care. Such an approach is conducive to achieving sustainable development goal 3 to "ensure healthy lives and promote well-being for all at all ages." 16 Despite the important role to be played by home-based records, there are a limited number of studies of the management of home-based records and most are focused on child vaccination cards. Nevertheless, some challenges to setting up effective and efficient home-based records have been identified. First, ensuring efficient, sustainable supplies of records is needed. Studies have highlighted the vulnerability of financing for home-based records; 17 poor stock management of records at national, provincial, district and facility levels; 18 and fragmented and parallel operations of multiple records. 19 Second, the availability of and access to home-based records differs between and within countries. 2 Sub-national analyses of the management of home-based records find that the availability of and access to these records differs between low-and high-income groups, 20 between rural and urban areas, 21 and between less and more educated mothers. 21 Third, studies have reported misuse or poor use of home-based records by both service users (such as pregnant women, mothers and other caregivers) and service providers (such as health-care workers). For instance, mothers who are indifferent to home-based records are likely to damage, misplace or lose the records. 2 Some health-care workers are careless about recording or reluctant to record the results of health check-ups, diagnosis and treatment in the homebased record. 9 The question why misuse or inadequate use of home-based records takes place has not yet been clearly answered. We hypothesize that poor use of homebased records might be attributable to a mismatch between their expected functions and the reality of users' capacities to manage the records. One study on the diverse functions of home-based records discussed three possible functions of child vaccination cards: (i) to increase mothers' knowledge about and demand for health-care services; (ii) to facilitate communication between mothers and health-care workers; and (iii) to reduce missed opportunities for health-care services. 22 The lack of studies on the relationship between the functions of home-based records and users' capacities makes it difficult to devise strategies for optimal use of such records. We propose that the users of home-based records need a specific set of capacities to enable the expected functions of the records to take place. WHO's recent guidelines, although a welcome initiative, recommend appropriate use of home-based records without also emphasizing the relationship between expected functions and users' capacities. 1 In this paper, we summarize the expected functions of home-based records and propose a function-capacity conceptual framework for home-based records.
Functions of home-based records We reviewed earlier studies on the operation of different types of home-based records, to explore differences and similarities in their expected functions. We conducted a non-systematic review of materials, including references listed in the WHO guidelines, documents generated by respective governments for their records, and data in the public domain. Table 1 summarizes the range of functions for home-based records as reported in earlier studies. We categorized the expected functions into three levels: data recording and storage (level 1); behaviour change communication (level 2); and monitoring and referral (level 3). Both types of homebased record users (i.e. health-care service users and providers) potentially benefit from all three levels of functions of home-based records. First, data recording and storage (level 1) is, by definition, the minimum common function of all home-based records. Home-based records make personal data related to maternal and child health care readily available and accessible at home. Home-based records can be taken to and used in different facilities, whereas facility-based records are attached to a specific facility. When data are appropriately recorded, the likelihood that home-based records serve as the reliable documented source of individuals' health data increases. 35 Those individuals' health and biological data can be used not only for health care, but also for other relevant purposes. Some home-based records are designed to serve as the birth certificate, and sometimes even as a reference for local civil registration systems, such as in Burundi. 30 This function helps home-based records serve as the documented evidence for home-based record owner's eligibility for subsidized health and other social services, such as free maternal and child health services according to child's age. 30 Home-based records can be designed to serve as one of the data sources for performance assessments of health-care workers or health facilities, particularly when connected to performance-based financing. 30 By double-checking children's immunization histories recorded in home-based records, over-vaccination can be avoided. 37 Home-based records often function as a reliable source of data for health surveys and research, for example, for estimating essential programme coverage or confirming the reliability of estimates. 36, 38, 39 Second, behaviour change communication (level 2) is expected in certain types of home-based records, typically in integrated, rather than stand-alone records. Health guidance pages embedded in records help mothers to practise home-based self-learning and peer education for taking necessary action on maternal and child health-related issues. The pages also help health-care workers to deliver educational messages more effectively. The level 2 function of home-based records enables mothers to be equipped with knowledge about danger signs and appropriate lifestyle changes during pregnancy and child care. Mothers can be given guidance on the appropriate timing of services, such as vaccination schedules, and how to practise home-based care. 7,20,22,23,25,2 6,29,31,39,41,42 Personalized guidance, such as upcoming service appointments, can be recorded by health-care workers. The function of triggering family members' positive behaviour changes was reported in several earlier studies on integrated home-based records. For example, fathers may be encouraged to quit smoking, make financial preparations for their baby's delivery, pay attention to keeping the newborn appropriately warm and increase interaction with their children to benefit early child development. 7, 8, 27 A key function at level 2 is supporting health-care workers in effectively conveying health messages to mothers and caregivers. For example, the home-based record can provide guidance on the respective stages of maternal and child health care and on seamless use of services across the continuum of care. 22, 23, 25, 29, 40, 41 Third, monitoring and referral in home-based records (level 3) enables health-care workers to correctly and efficiently track the personal health data and treatment histories of clients. This function is important for maternal and child health services because pregnant women and mothers usually receive
Functions of home-based records by level and user Level 1 function: data recording and storage For target beneficiary Makes personal reproductive and maternal data (on pregnancy, childbirth, postpartum and between pregnancies) readily available and accessible at home, and more mobile 5, 22 Makes personal child immunization data readily available and accessible at home, and more mobile 3, 23 Makes personal childhood anthropometric data in a line chart readily available and accessible at home, and more mobile 4 Makes personal data at all stages of maternal and child health (pregnancy, childbirth, postpartum and newborn care; child immunization, growth and development) readily available and accessible at home, and more mobile 20, 21, Often serves as the documented evidence for eligibility for free or subsidized health-care and other social services, and for assessing performance of results-based financing 30,35 (continues. . .)
Policy & practice Home-based records and users' capacities Keiko Osaki & Hirotsugu Aiga
Characteristics and functions of records
Type of home-based record
Maternal health card or handbook
Child vaccination card
Growth chart
Maternal and child health handbook For health-care workers Serves as the reliable source of individuals' reproductive and maternal data for appropriate clinical decisions 22 and for health statistics, surveys and research 22 Serves as the reliable source of individual children's immunization data for clinical decision-making (e.g. avoiding missed vaccination opportunities) 23 and for health statistics, surveys and research. 36 Saves costs related to unnecessary vaccinations through double-checking children's previous immunization history on the card 23, 37 Assists health-care workers to detect child malnutrition and intervene in a timely way 4 Increases clinical efficiency by allowing health-care workers to check records and avoid unnecessary repetition of questions, examinations and vaccinations on maternal and child health. 19 Serves as the reliable, comprehensive source of individuals' maternal and child health data for health statistics, surveys and research 36, 38, 39 Level 2 function: behaviour change communication For target beneficiary Equips pregnant women with knowledge about danger signs and lifestyle changes, to promote selfmanagement during pregnancy, postpartum and between pregnancies. 22 Often guides pregnant women and family members on upcoming care visit appointments of maternal services 7, 9, 22, 23, 26, 29, 32, 33, 35, 38, 43 Clients' strategic choice of facilities according to their own health-care needs is increasingly common not only in high-income countries but also, more recently, in low-and middle-income countries. 28, 32 Even those women who originally planned to use maternal and child health services at a single facility can benefit when the home-based record serves as a referral letter to other facilities. Some home-based records require mothers and caregivers to record the results of self-monitoring before and after service appointments. This allows health-care workers to attend to maternal and child health-related issues that are observable only at home, such as child feeding, child development or maternal depression. 8, 27, 35, 45 The level 3 function aims to promote continuous self-monitoring, thereby empowering mothers and caregivers to recognize and address health risks via self-care or self-referral to a higher or lower level of health facility. 8, 20, 32, 33, 43 The process of self-monitoring and self-recording on child development milestones can provide mothers and caregivers with an opportunity to increase interaction with and attachment to their children. 27, 46
Function-capacity framework Categorizing the various functions of home-based records raises the question whether and to what extent the users are knowledgeable and skilful enough to make the expected functions happen. More importantly, what capacities do users need to realize these functions? This question has rarely been raised, despite its importance as a critical determinant of the type of home-based record to be employed. We assume that different levels of home-based record functions require users to be equipped with different types and levels of capacities. Fig. 1 illustrates the hypothetical capacity requirements of users of home-based records in relation to the three levels of function that we have outlined. Note that users' capacities here encompass not only knowledge and potential abilities, but also motivation and attitudes that enable practices.
Characteristics and functions of records
Type of home-based record
Maternal health card or handbook
Child vaccination card
Growth chart
Maternal and child health handbook For target beneficiary Promotes self-monitoring of maternal health status during pregnancy, postpartum and between pregnancies. 43 Increases mothers' self-control over pregnancy. 43 Empowers pregnant women to recognize risks and to self-refer to a higher or lower level health facility 22 Promotes self-monitoring of children's vaccination status by mothers or other caregivers, for better self-planning for upcoming child immunization visits. 23 Increases mothers' or other caregivers' sense of ownership over their children's health 23 Provides mothers and other caregivers with opportunities to self-monitor their children's nutritional status 42 Promotes overall and continuous self-monitoring of maternal and child health status by mothers and other caregivers. For health-care workers Serves as the means of promoting continuity of care throughout a woman's reproductive life. 22 Empowers community health-care workers to recognize risks and, if necessary, refer mothers to a higher level health facility. 22 Serves as the referral form to a higher or lower level health facility along with chronological data of current pregnancy 22 Serves as the reliable source of child's individual vaccination data to be shared across different health facilities 23 NA Serves to promote a continuum of care throughout all stages of maternal and child health care. 7,9,26,28,29,33,35,38,43 Serves as a personalized checklist that enables healthcare workers to adhere to national clinical protocols. 24 Serves as the referral form to a higher or lower level health facility along with chronological data of maternal and child health 32, 33, 44 NA: not applicable.
(. . .continued) First, to keep a home-based record functioning as a data recording and storage tool, users of the records need only minimum capacities. Health-care workers need to be capable of recording the results of health-care services in the records. Mothers and caregivers need to be capable of retaining the records at home without damaging, misplacing or losing them and to take them to the points of services. The contents of home-based records need not be fully understood by mothers and caregivers at this level of function. However, it is not clear to what extent home-based records are functioning as data recording and storing tools in low-literacy settings. WHO has indicated that poor retention of home-based records by mothers and caregivers needs to be addressed, while acknowledging that some countries have achieved success in home-based record retention. 1 Loss of home-based records was reported as the critical issue by several earlier studies too. 2, 3 A low level of literacy among mothers is one factor that could affect receipt and loss of home-based records by mothers, although factors unconnected to users capacities, such as poorly functioning health systems, could also affect the use of these records. 1 Second, for home-based records to function as a behaviour change and communication tool, mothers and caregivers need greater capacities beyond literacy. To enable a home-based record to trigger behaviour change through self-learning and peer-education, mothers and caregivers should be able not simply to read and understand the contents of guidance pages, but also to incorporate them into practice, when needed, with the help of other family members. Health-care workers should not simply be literate, but also technically skilled enough to translate the information into the local maternal and child health context. Therefore, healthcare workers need to provide guidance to women about the content and value of the home-based record. Third, if home-based records are to function as a monitoring and referral tool, health-care workers need to be equipped with the knowledge and skills to use the records for appropriate, comprehensive clinical decisions. This needs to happen at all levels of health care (primary, secondary and tertiary) within maternal and child health programmes and in both the public and private sectors. Mothers and caregivers need to present the record at different facilities to allow health-care workers to refer to and update the data and hence leverage the inter-facility mobility of home-based records. To record data on child development, mothers and caregivers need the capacity in their daily routine to objectively observe their children's behavioural and cognitive responses against the child development milestones in home-based records. 45
Filling the capacity gap Fig. 1 assists health policy-makers in identifying the discrepancy between the functions a health system demands and the functions that can be realized within the capacities of current users. When a home-based record designed for a lower-level function is employed in a setting with users of higher capacities, health policy-makers can, in theory, expand the functions to cover the higher levels. On the other hand, when a record designed for higher-level functions is employed in settings with users of lower capacities, policy-makers need to downgrade functions or add supplementary elements to the record and its operation. Typical supplementary elements include designing records for greater user-friendliness and adding supportive interventions for increasing the capacities of record users. Accurate understanding of users' current capacities will inform a more user-friendly and effective design. Some health-care workers are not motivated to record clinical data in home-based records because they see the task as an additional workload. 3 To encourage recording, the layout of entry columns in a home-based record should be carefully designed to enable selected clinical data to be efficiently and correctly transcribed from facility-based records to, for example, the maternal and child health logbook or child immunization logbook. 47 Losing and not receiving home-based records are concerns. 2 Reports from Indonesia, Kenya, Uganda, and West Bank and Gaza Strip suggest that records with graphics and illustrations were more understandable and attractive to illiterate or less literate mothers and might have contributed to higher coverage and lower loss of homebased records. 44 More user-friendly design is effective, particularly when the health system requires level 2 functions, but users' capacities remain at the level 1 function. Some home-based records include vouchers for immunization services or food rations as in Japan. 10, 35 Embedding incentive vouchers in records often increases a sense of ownership and hence retention of home-based records by mothers and caregivers. This approach is effective when users may not have the capacity to fulfil level 1 func-
Fig. 1. Hypothetical progression of users' capacities that enable home-based records to function as designed Increasing capacities of health-service providers Increasing capacities of health-service users Remind users to take home-based records to higher or lower level facilities. Recognize home-based records as the referral form. Use home-based records for health education. Conduct clinical tasks. Record data in home-based records. Retain home-based records at home. Take home-based records to health facilities at visits. Read and understand home-based records. Record maternal and child health care status. Take home-based records to health facilities at referral.
& practice Home-based records and capacities Keiko Osaki & Hirotsugu Aiga tions. Minimizing the use of medical terms and avoiding wordiness in the texts increases the feasibility of designing home-based records for higher-level functions. 12, 44 Supportive interventions for increasing users' capacities and orientation and refresher training for health-care workers are essential for realizing all levels of home-based record functions. 1 The contents of training for health-care workers could be designed to fill the gaps between current capacities and required capacities. For instance, in-service training on health education methods are an effective means of increasing the feasibility of level 2 functions in homebased records. 48, 49 Training during preservice education could be an efficient strategy that pre-empts a need for filling gaps in health-care workers' capacities. In some countries, health professional associations award specialist accreditation to those who have practised quality care including health education by using home-based records. Training programmes conducted by nongovernmental organizations could help ensure nationally standardized home-based records are used in both public and private health facilities. 15, 28 Some countries found that follow-up supportive supervision reinforces the capacities of health-care workers. 46 Particularly when training programmes are not readily available and accessible for health-care workers; guides or manuals serve as practical tools that assist health-care workers towards smoother and more efficient use of records. 34, 50 Health-care workers' guides are often developed at the time of development or revision of home-based records. 33
Conclusion To accommodate the growing demands of national health policies and health systems, policy-makers tend to make overambitious plans for home-based records. As a result, important functions of these records often remain underused, leading to loss of confidence in the records and to a waste of resources and opportunities for care. There are several key capacities of home-based record users that act as determinants of optimal use of home-based records. At a minimum level, mothers and caregivers should be capable of carefully retaining homebased records and taking them to the points of service. For higher levels of functions, mothers need the capacities to translate maternal and child healthrelated messages into their own context and take sensible decisions about actions over time. Health-care workers need the basic capacity to undertake stock management of records and to record data in records. At higher levels of function, they need the capacity to explain the data and health-related messages to mothers and caregivers and to translate these for appropriate clinical decisions. When designing a home-based record, the capacities of both types of users must be assessed and considered for optimizing the use of the records. Today, several international guides and manuals on home-based records have been published by WHO. 1, 3, 5 However, these guides do not fully address ways of strategically selecting the most suitable home-based record for each country, in accordance with the capacities that users currently have or potentially would have. One reason for this may be an absence of evidence that identifies the relationship between the level of home-based record functions and users' capacities. Mismatches between the expected functions of records and the realities of users' capacities need to be examined for the optimal use of home-based records in maternal, newborn and child health care. Competing interests: None declared.
摘要
Resumen Adaptación de los registros domiciliarios sobre salud materno-infantil a las capacidades de los usuarios Los registros domiciliarios se han utilizado tanto en países de bajos ingresos como en los de altos ingresos para mejorar la salud maternoinfantil. Tradicionalmente, se trataba en su mayoría de registros independientes que apoyaban un único programa relacionado con la salud materno-infantil, como la tarjeta de vacunación infantil o el gráfico de crecimiento. Hace poco, un número cada vez mayor de países ha empezado a utilizar registros integrados domiciliarios para apoyar todos o una parte de los programas relacionados con la salud materno-infantil, como en el manual de salud materno-infantil. Las expectativas de los responsables de la formulación de políticas con respecto a los registros domiciliarios son a menudo demasiado ambiciosas y muchas funciones importantes de los registros no se aprovechan, lo que conduce a una pérdida de confianza en el proceso y a un despilfarro de recursos y oportunidades de atención. Es necesario examinar las brechas entre las funciones de los registros y la medida en que los usuarios de los mismos (mujeres embarazadas, madres, cuidadores y trabajadores de la salud) tienen el conocimiento y la habilidad suficientes para que esas funciones se cumplan. Se pueden planificar tres funciones clave, con niveles crecientes de complejidad, en los registros domiciliarios: (i) registro y almacenamiento de datos, (ii) comunicación de cambios de comportamiento, y (iii) seguimiento y remisión. Se ha definido un marco conceptual de función y capacidad para los registros domiciliarios que muestra cómo un número y una complejidad crecientes de las funciones en un registro domiciliario requiere una mayor capacidad de
Policy & Home-based records and users' capacities Osaki & Hirotsugu Aiga sus usuarios. El tipo y las funciones de un registro domiciliario óptimo deben seleccionarse estratégicamente de acuerdo no solo con las demandas del sistema de salud, sino también con las capacidades de los usuarios del registro. Table 1 . 1 Characteristics and functions of home-based records for maternal and child health , home-based record: specific to reproductive and maternal health programme Stand-alone, home-based record: specific to expanded programme on immunization Stand-alone, home-based record: specific to child nutrition programme Integrated, home-based record: all maternal and child health stages Document style One-page card, often foldable; or 20-30 page bound booklet One-page card, often foldable One-page card, often foldable 40-60-page bound booklet Target beneficiary Pregnant women and mothers Children Children Pregnant women, mothers and children
7-10,28,29,33,35,43 Increases self-control over maternal and child health that leads to greater satisfaction and self-decision-making. 8,43 Empowers mothers to recognize risks and to self-refer to a higher or lower level health facility 8,20,33,43 Promotes interaction and thereby attachment between mothers or caregivers and their children through self- monitoring child development 7,27,33,35
ملخص املستخدمني قدرات مع والطفل األم لصحة املنزلية السجالت مواءمة 博和技术娴熟程度之间的差距 以实现预期功能。随 益增加的功能数量和复杂性程度如何要求其使用者具 着复杂性的增加 , 可以在基于家庭的记录中规划三项 备更强大的能力。基于家庭的最佳记录类型和功能不 关键功能 :(i) 数据记录和存储 ;(ii) 行为改变沟通 , 以及 仅应根据卫生系统的需求 , 而且应综合记录使用者所 (iii) 监测和转诊。我们为基于家庭的记录定义了一个 具备的能力进行战略性选择。 功能--能力概念框架 , 显示了基于家庭的记录中 , 日 Résumé واألمهات احلوامل )النساء السجالت مستخدمي ومهارة دراية مما كاف بقدر الصحية( الرعاية يف والعاملني الرعاية ومقدمي وظائف لثالث التخطيط يمكن الوظائف. هذه تفعيل يف يسهم التعقيد: يف متزايدة مستويات ذات املنزلية، السجالت يف رئيسية السلوك، تغيري تواصل و)2( وختزينها؛ البيانات تسجيل (1) للقدرة مفاهيمي عمل إطار نحدد نحن واإلحالة. واملراقبة و)3( والتعقيد املتزايد التعدد أن يوضح مما املنزلية، للسجالت الوظيفية مستخدميها. بني أكرب قدرة يتطلب املنزلية، السجالت وظائف يف بشكل ووظائفه املثايل املنزيل السجل وظائف نوع اختيار جيب ولكن الصحي، النظام مطالب مع فقط ليس يتوافق، بام اسرتاتيجي السجالت. مستخدمي قدرات مع أيضا الدخل ذات البلدان من كل يف املنزلية السجالت استخدام تم نحو وعىل والطفل. األم صحة لتحسني املرتفع والدخل املنخفض ًا برناجم تدعم بذاهتا قائمة سجالت الغالب يف هذه كانت تقليدي، أو الطفل تطعيم بطاقة مثل والطفل، األم بصحة متعلق ا واحدً البلدان من متزايد عدد يستخدم ، ً وحديثا به. اخلاص النمو خمطط املتعلقة الربامج من جزء أو كل لدعم املتكاملة املنزلية السجالت ما ً وغالبا والطفل. األم صحة كتيب يف كام والطفل، األم بصحة يف مفرطة املنزلية السجالت من القرار صناع توقعات تكون مما مستغلة، غري للسجالت اهلامة الوظائف تزال وال الطموح، الرعاية. وفرص املوارد وإهدار العملية، يف الثقة فقدان إىل يؤدي ومدى السجالت وظائف بني الفجوات دراسة إىل بحاجة نحن 根据使用者能力调整基于家庭的孕产妇和儿童健康记录 低收入和高收入国家均使用基于家庭的记录来改善孕 和儿童健康相关的计划 , 例如母婴健康手册。政策制 产妇和儿童的健康。传统上 , 这些独立记录大多是支 定者对基于家庭的记录往往期望过高 , 而不能充分运 持与孕产妇和儿童健康相关的单一记录 , 例如儿童疫 用其重要功能 , 导致对该进程失去信心 , 并浪费护理 苗接种卡或儿童生长表。最近 , 越来越多的国家正在 资源和机会。我们需要检查记录的功能与记录使用者 使用基于家庭的综合记录来支持所有或部分与孕产妇 ( 孕产妇、母亲、看护人和医疗保健工作者 ) 的知识渊
Adapter les fiches de santé de la mère et de l'enfant tenues à domicile aux capacités des utilisateurs Les fiches de santé tenues à domicile sont aussi bien utilisées dans des pays à revenu faible que dans des pays à revenu élevé pour améliorer la santé de la mère et de l'enfant. Habituellement, les fiches utilisées étaient essentiellement des documents indépendants, associés à un programme spécifique de santé de la mère et de l'enfant, tels que les carnets de vaccination ou les courbes de croissance des enfants. Depuis quelque temps, un nombre croissant de pays opte pour des fiches plus complètes, couvrant tout ou partie des programmes de santé de la mère et de l'enfant, comme les manuels de santé de la mère et de l'enfant. Les attentes des décideurs politiques autour des fiches de santé tenues à domicile sont souvent trop ambitieuses, et des fonctions importantes des fiches sont sous-employées, ce qui rend le processus moins fiable, gâche des ressources et fait perdre des opportunités de soins. Il est nécessaire d' examiner l' écart entre les fonctions des fiches et le degré de connaissance et de compétence de leurs utilisateurs (femmes enceintes, mères, aidants, professionnels de santé) pour véritablement tirer parti de toutes les fonctions prévues. Trois principales fonctions, énoncées dans l'ordre croissant de leur degré de complexité, doivent être prévues dans les fiches de santé tenues à domicile: (i) enregistrement et stockage des données; (ii) communication des changements des comportements et (iii) suivi et recommandations pour le parcours de soins. Nous avons défini un cadre conceptuel Fonction-Capacité pour les fiches de santé tenues à domicile, qui montre comment des fonctions plus nombreuses et plus complexes nécessitent de plus grandes capacités chez les utilisateurs. Le type et les fonctions d'une fiche de santé optimale devraient être choisis stratégiquement en fonction, non seulement des exigences des systèmes de santé, mais aussi des capacités des utilisateurs des fiches. Резюме
Адаптация хранимых на дому записей о здоровье матери и ребенка под способности пользователей Хранимые на дому записи о здоровье используются в странах как пользователи таких записей (беременные женщины, матери, с высоким, так и с низким уровнем дохода для совершенствования лица, осуществляющие уход, и работники здравоохранения) системы охраны здоровья матери и ребенка. Традиционно обладают навыками и знаниями, необходимыми для реализации это были в большинстве своем отдельные записи, которые ожидаемых целей. Можно запланировать три основных способствовали поддержке единой программы охраны мероприятия (с возрастающим уровнем сложности) по здоровья матери и ребенка, например карта вакцинации или ведению хранимых на дому записей о здоровье: (I) ведение карта физического развития. С недавних пор все большее записей и их хранение; (ii) просветительская работа в целях количество стран используют хранимые на дому записи в изменения моделей поведения; (iii) мониторинг и направление качестве элемента полной или частичной поддержки программ к специалистам. Авторы определили концептуальную модель по охране здоровья матери и ребенка, например руководство целей и способностей применительно к хранимым на дому по охране здоровья матери и ребенка. Часто ожидания тех, кто записям о здоровье, которая демонстрирует, что увеличение принимает стратегические решения, по поводу таких хранимых количества целей хранимых на дому записей и повышение на дому записей оказываются чрезмерно завышенными, а важные их сложности требует совершенствования способностей функции записей используются не в полной мере, что приводит пользователей. Тип и цели оптимальной системы хранимых на к утрате доверия к процессу, напрасной трате ресурсов и дому записей о здоровье должны определяться стратегически возможностей получения медицинской помощи. Необходимо с учетом не только потребностей системы здравоохранения, но изучить расхождения между целями записей и тем, в какой мере и умений и навыков пользователей записей.
|
10.1016/j.elecom.2022.107424
|
cc-by
| null | null |
openalex
|
This article offers insight into the role of binders in the overall performance of a dual-ion battery (DIB). Replacing sodium carboxymethyl cellulose (CMC) with poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP) enhances the interfacial stability of a graphite positive electrode in a DIB. Electrochemical testing combined with Xray photoelectron spectroscopy (XPS) and operando pressure measurements highlight that PVdF-HFP suppresses parasitic reactions at the cathode-electrolyte interface (CEI), in sharp contrast with CMC. However, CMC causes less interfacial resistance and is hence beneficial in terms of rate capability.Introduction The graphite dual-ion battery (GDIB) is an emerging technology for stationary energy storage, with a unique operational mechanism entailing anion intercalation into a graphite cathode . This feature translates into a cheap, safe and environmentally-benign cell chemistry, due to the elimination of transition metal oxides. Furthermore, capacities in the order of 80-100 mAh g -1 [2, 3] combined with a high cell voltage (~4.5 V) result in an energy density suitable for stationary applications. Be that as it may, the high potentials required for anion intercalation in graphite cause electrolyte breakdown and Al current collector corrosion . Parasitic reactions can be partly minimized when highly concentrated electrolytes (HCEs) and ionic liquids (ILs) are employed . High salt concentrations increase electrolyte stability via extensive solvent coordination towards Li + [8, 9] and suppress Al dissolution in the form of Al 3+ -anion complexes [10, 11] . Nonetheless, HCEs provide only kinetic stabilization and do not entirely prevent side-reactions. In state-of-the-art Li-ion technologies, the negative and positive electrodes are usually protected by functional solid-electrolyte interface (SEI) and CEI layers, respectively, which ensure coulombic efficiencies (CEs) > 99 % and long cycle life. The SEI formed on graphite anodes cycled in electrolytes based on LiPF 6 and carbonate solvents is unarguably the most well-studied interphase and a main contributor to the commercialization of LIBs . On the contrary, CEI-studies on graphite cathodes remain still scarce . Apart from electrolyte additives, functional binders that can enhance the stability of the SEI [22, 23] or CEI layer are of interest for DIBs. Binders resulting in adequate active material surface coverage can suppress solvent decomposition and mitigate solvent cointercalation which, in graphite, causes destructive exfoliation . In this regard, few studies have focused on binders for graphite cathodes in DIBs. The in situ dilatometric study conducted by Huesker et al. showed that coatings with CMC expanded to a greater extent than PVdF-based ones and led to particle detachment upon extended cycling . Chan et al. examined the binder-dependent power characteristics of GDIBs. CMC-based graphite cathodes outperformed PVdF-based ones in rate capability tests (83 mAh g -1 vs 43 mAh g -1 at 500 mA g -1 ), leading to the conclusion that fluorinated binders increase the interfacial resistance . This work explores the relationship between binders and CEI stability. Two commonly used binders, CMC and PVdF-HFP, are studied using galvanostatic cycling, electrochemical impedance spectroscopy (EIS), Raman spectroscopy, operando pressure monitoring and X-ray photoelectron spectroscopy (XPS). It is revealed that, despite being viewed upon as an inactive component, the binder does substantially affect the interfacial stability and self-discharge properties in GDIBs.
Experimental Electrode preparation. Cathodes were prepared by mixing 90 wt% natural graphite (Ted Pella) with either 10 wt% CMC (Leclanché) in a 9:1 water: ethanol solution or with 10 wt% PVdF-HFP (Arkema) in Nmethyl-2-pyrrolidone (NMP). Mixing took place in a Retsch PM400 shaker (30 min at 25 Hz) and slurries were cast on carbon-coated Al-foil. The mass loadings of CMC-and PVdF-HFP based electrodes amounted to 1.4 and 2.0 mg cm -2 , respectively. Electrodes were dried under vacuum at 120 C for 12 h prior to use. Electrolyte preparation. Dimethyl carbonate (DMC, Sigma Aldrich) was dried over activated molecular sieves (3 Å) for 48 h and filtered through a 200 nm PTFE membrane. Lithium bis(fluorosulfonyl)imide (LiFSI, 99.9 %, Solvionic) was dissolved in DMC to prepare a 4 M concentrated electrolyte. Electrochemical characterization. Cycling took for the most part place in two-or three-electrode Li-graphite pouch-cells with a glassfiber separator impregnated with 150 μL of 4 M LiFSI in DMC. Galvanostatic cycling for ex-situ characterizations was performed at 20 mA g -1 , between 3.0 and 4.9 V vs Li + /Li, using a Neware battery cycler. Rate tests were performed at 50, 100, 200 and 500 mA g -1 . An MPG Biologic potentiostat was used for self-discharge and EIS tests. Spectra were collected in a three-electrode setup, for frequencies between 20 kHz and 10 mHz and with a 5 mV amplitude. Spectra were collected 1) prior to cycling, 2) at the charged state (at 4.9 V vs Li + /Li), 3) after a 24 h opencircuit voltage (OCV) step and 4) after a constant-current constantvoltage (CCCV) discharge step (3.0 V vs Li + /Li). SEM/EDS. Micrographs were acquired with a Zeiss Merlin Microscope at 3.0 kV acceleration voltage, 100 pA probing current and 6.7 mm working distance. EDS maps were acquired with an X-Max 80 mm 2 silicon drift detector, at 6.0 kV, 500 pA and 8.5 mm. X-ray photoelectron spectroscopy. Cycled electrodes were rinsed with DMC, dried in the glovebox and transferred inertly to a Kratos Axis Supra+ spectrometer equipped with an Al anode. Acquisitions took place in floating mode with charge compensation and with a 15 mA emission current. The data was fitted in IgorPro after subtraction of a Fig. 1 . In a) and b) voltage-capacity profiles of electrodes based on CMC and PVdF-HFP, respectively. In c), discharge capacity and coulombic efficiency data from the rate capability test. Plot d) shows the first cycle for CMC and PVdF-HFP electrodes charged to 4.9 V at 20 mA g -1 and subjected to a 24 h self-discharge test. The numbering corresponds to distinct points on the cycling curve, at which EIS measurements were conducted: 1. charge, 2. 24 h self-discharge, 3. discharge and 4. after a CV-step following the discharge. The EIS spectra for these four different conditions and for the three first cycles are shown in e) for CMC and in f) for PVdF-HFP. Shirley background and calibrated using the graphite C 1 s peak (284.0 eV). Raman spectroscopy. A Renishaw inVia Ramanscope with a 50-mW 532 nm laser was employed to collect Raman spectra. A 50 X objective lens, 5 % laser power and 20 s exposure time were used during measurements and a minimum of 8 spots were analyzed per sample. Operando pressure monitoring. Cell pressure was monitored by running measurements in a three-electrode PAT-cell (Li 4 Ti 5 O 12 (LTO)graphite with a Li reference), using the same cycling protocol as in the self-discharge tests. Cells were cycled in a climatic chamber at 30.4 °C.
Results and discussion The rate test results are presented in Fig. 1a-c . Electrodes with PVdF-HFP delivered higher discharge capacity (77 mAh g -1 ) at low specific currents (20 mA g -1 ) compared to CMC (67 mAh g -1 ). At specific currents of 500 mA g -1 , the capacity of the PVdF-HFP electrode dropped to 53 mAh g -1 , while that of CMC increased (72 mAh g -1 ). The capacity decrease for PVdF-HFP was probably related to anion diffusion limitations across the CEI. The steady performance of CMC at high rates indicated favourable kinetics and low interfacial resistance, as previously reported . At 20 mA g -1 , electrodes with CMC exhibited a poor initial coulombic efficiency (iCE) of 63 %, which increased to 80 % over 5 cycles. PVdF-HFP electrodes exhibited an iCE of 83 % at the same rate, which subsequently rose to 96 %. The low CE originates primarily from electrolyte-consuming side-reactions on the graphite cathode and corrosion of the Al current collector. Aggravated degradation is observed at slow cycling rates which allow sufficient time for parasitic reactions to occur. Evidently, increasing the current to 500 mA g -1 improved the reversibility of anion intercalation with the CE reaching 99 % for CMC and 97 % for PVdF-HFP. The binder affected the anion intercalation overpotential as well. Differential capacity (dQ/dV) curves (Fig. S2 & S3 ) showed that the initial intercalation onset voltage was located at 4.31 V for PVdF-HFP and 4.32 V for CMC. In both cases, a shift towards lower values was noted in the second cycle: 4.29 V for PVdF-HFP and 4.28 V for CMC. This is linked to the graphite activation, including interlayer expansion and improved electrode wetting . When the current increased to 500 mA g -1 , the intercalation potential increased from 4.32 to 4.37 V for PVdF-HFP and from 4.29 to 4.31 V for CMC. Interestingly, the largest impact was observed during deintercalation; while the dQ/dV curves for CMC remained fairly similar, the overpotential for PVdF-HFP increased by almost 0.4 V when the current rose from 20 to 500 mA g -1 . The worse performance shown by PVdF-HFP binder, after the rate test, was attributed to anion trapping, as discussed later on (Fig. 3a ). Such a limiting effect was not observed for long-term cycling at low specific currents (see Fig. S4 ). The binder influenced the self-discharge behavior of anionintercalated graphite electrodes (Fig. 1d ). Upon relaxation at opencircuit for 24 h, the potential of a charged graphite cathode with CMC binder dropped from 4.9 to 4.28 V, while that based on PVdF-HFP stabilized at 4.72 V. In the subsequent discharge process, only 12 % of the expected charge could be extracted from the CMC-electrode, as opposed to 50 % for PVdF-HFP. The lower self-discharge observed in the case of PVdF-HFP could be caused by several factors, including its own oxidative stability, as well as its ability to suppress the contact between the charged graphite cathode and electrolyte components that could be oxidized (e.g. the solvent). EIS measurements on three-electrode cells indicated differences in intercalation overpotentials in the presence of the two binders. In particular, the CMC-based electrode exhibited a lower charge transfer resistance as compared to the one containing PVdF-HFP (compare Nyquist plots marked "charge" and "OCV" in Fig. 1e-f ). In addition, indications of differences in anion trapping effects were suggested by the "discharge" and "CV" EIS spectra. As the imaginary (capacitive) impedance contribution appeared to be larger in the case of CMC-based electrodes, it was assumed that anion trapping was reduced as opposed to PVdF-HFP (Fig. 1e-f ). XPS measurements on pristine and cycled electrodes (discharged to 3.0 V vs Li + /Li) highlighted the binders' influence on the evolution of the cathode surface chemistry (Fig. 2 ). The C 1s spectra of pristine CMCbased electrodes indicated good surface coverage of the graphite particles, as the -C-H/-C-C-(sp 3 -bonded carbon), -C-O-, -O-C-O-and -C(=O)O -signals were prominent at 285.0, 286.9, 288.4 and 289.6 eV. The less intense peak from sp 2 -hybridized carbon at 284.0 eV further confirmed that graphite particles were initially chiefly buried underneath the CMC binder. In the O 1s spectra, the peaks at 533.3 and 531.6 eV corroborated the presence of -C -Oand C--O groups, respectively, while minor signals at 534.5 and 536.3 eV were assigned to C-O-H and Na KLL Auger peaks. In pristine electrodes containing PVdF-HFP the -C-H, -CH 2 CF 2 , -C-F, -CH 2 CF 2 and -CF 3 binder functionalities appeared at binding energies of 285.0, 286.0, 289.2, 290.7 and 293.5 eV, while -C-Osurface groups were still detectable at ≈ 287 eV. Still, the most intense contribution to the C 1s signal stemmed from sp 2 -bonded carbon (284.0 eV), suggesting that PVdF-HFP was not homogeneously distributed around the graphite flakes. This is in good agreement with previous literature, as PVdF-HFP has proven to preferentially adsorb onto the defective graphite edges . Surface oxygen was detected at 531.8 and 533.3 eV in the O 1s spectra, while the fluorine signal from PVdF-HFP was primarily located at 687.7 eV. Exposing CMC-based electrodes to the 4 M LiFSI in DMC electrolyte (OCV sample) led to differences in the C 1s and O 1s spectra (compared to the pristine sample), which were associated to electrolyte uptake. Notably, the -C -H signal increased for the OCV sample, indicating that DMC trace amounts might remain trapped in the electrode. O 1s signals due to DMC/ FSI traces were also observed. Peak splitting was noted in the F 1s spectra: the fluorine in FSI was detected at a binding energy of 687.9 eV, while a secondary peak at 689.7 eV is suspected to be an electrolyte impurity. The 'OCV' electrode with PVdF-HFP exhibited similar changes to the CMC-based one, due to the contributions of the electrolyte to the C 1s, O 1s and F 1s spectra. Upon cycling, further changes incurred in both systems. In CMCbased electrodes, the -C -O-, -O -C -O-and -C(=O)O-signals decreased in relative intensity compared to the graphite substrate. In fact, the graphite signal was most intense in the C 1s spectra after the 125th discharge, indicating that the organic component of the CEI layer (herein including contributions from the binder and electrolyte decomposition products) was thin and/or non-homogeneously distributed. The consistent decrease in binder-related signals in the C 1s corelevel spectra is telling of partial CMC degradation, which may have its root in both electrochemical and mechanical processes. Furthermore, the appearance of an additional peak in the F 1s spectra of several samples (685.0-685.5 eV) indicated the growth of metal fluorides (possibly LiF and AlF 3 ) on the graphite cathode and Al current collector and hence suggested the partial decomposition of the FSI anion. On the contrary, cycled electrodes containing PVdF-HFP displayed an increase in C 1s signals linked to the binder/ decomposed electrolyte, indicating the formation of an organic CEI layer. Minor changes were noted in the F 1s spectra, indicating metal fluoride formation, alike that in the CMCbased electrodes. The graphite cathode morphology and binder distribution were tracked via SEM/EDS imaging (see Figs. S5-S8 ). In general, cycled graphite electrodes using either binder exhibited significant expansion, particularly visible at the flake edges. EDS mapping demonstrated the uniform distribution of CMC within both pristine and cycled electrodes, while some evidence of the selectivity of PVdF-HFP was presented as the F signal was most intense around the graphite particle contours. Ex situ Raman scattering analyses performed on the same, pristine and cycled samples (Fig. 3a ) provided information on structural changes in the graphite cathode. Pristine graphite electrodes with either binder formulation exhibited a high degree of crystallinity and structural order. This is deduced by the relative intensity of the first order scattering bands located at ≈ 1346 cm -1 (D-band) and ≈ 1580 cm -1 (G-band). The G-band is representative of the simultaneous bond stretching of sp 2 -bonded carbon atoms in the graphite rings, while the D-band reflects their respective breathing modes . In many cases, an intense D-band suggests the presence of defects, including stacking faults, edge structures, local stresses and graphene layer deformation . The first few charge-discharge cycles at low specific currents (20 mA g -1 ) did not affect the graphite flake structure considerably (Fig. 3a , cycle 1-10), as obtained spectra were reminiscent of the pristine graphite. Generally, the D'-band intensity increased slightly, while few samples (e.g., the electrodes extracted after the 10th discharge) showed evidence of incomplete discharge/ anion trapping. The latter phenomenon is tracked through the appearance of the additional D'-band, located at a slightly higher wavenumber as compared to the G-band (initially ≈ 1602 cm -1 ). The peak-splitting of the G-band is well-documented and is associated to the staging phenomenon and introduction of intercalants between graphene layers [32, 33] . Subjecting electrodes with either CMC or PVdF-HFP binder to the long-term rate test described in Fig. 1c did nevertheless point out substantial differences between the two systems (Fig. 3a , cycle 125). While a low degree of disorder was maintained for the CMC-based electrode, the graphite cathode with PVdF-HFP exhibited a substantial increase in both the D and D' bands, along with a shift of the D'-band towards higher wavenumbers (≈ 1617 cm -1 ). This is in line with previous observations: the persisting, more resistive CEI in electrodes with PVdF-HFP, impedes anion de-intercalation and induces trapping. Self-discharge experiments were in addition repeated in a setup allowing for operando pressure monitoring (Fig. 3b-c ). The pressure increase, ΔP, after two cycles amounted to 39 and 42 mbar in cells using electrodes with CMC and PVdF-HFP binder, respectively. After accounting for differences in the mass loading of the two coatings, ΔP is adjusted to 9.9 and 7.4 bar g -1 graphite for the CMC and PVdF-HFP systems, respectively. Any differences in the evolved gases would hence stem from the degradation of inactive components (e.g., the binder) and decomposition of electrolyte salt and solvent on the graphite cathode [15, 34, 35] . While no efforts were undertaken to identify the gaseous species here, previous findings suggest that carbonate solvent oxidation results predominantly in CO 2 and CO [34, 35] , while FSI decomposition yields SO x species . Ultimately, the comparatively larger (normalized) pressure increase observed in the case of CMC, further corroborates previous evidence questioning its capability to prevent the occurrence of parasitic reactions and graphite cathode self-discharge.
Conclusion In summary, this study sheds light on how binders impact the performance of graphite positive electrodes. Cycling at 20 mA g -1 resulted in higher iCE for PVdF-HFP (83 %) compared to CMC (63 %), suggesting aggravated side-reactions for the latter. Upon further cycling, the CE rose to 96 % for PVdF-HFP and 80 % for CMC, while the discharge amounted to 77 mAh g -1 and 67 mAh g -1 , respectively. High-rate cycling (500 mA g -1 ) led to decreased capacity for PVdF-HFP (53 mAh g -1 ), while an increase (72 mAh g -1 ) was recorded for CMC. High rates supressed side-reactions, resulting in increased CE for both systems (97 % for PVdF-HFP and 99 % for CMC). Overpotential trends complemented by EIS, indicated that PVdF-HFP formed a more resistive interface than CMC, which supressed side-reactions and self-discharge. Surface analyses and operando pressure measurements suggested worse CEI stability for graphite cathodes using CMC as opposed to those based on PVdF-HFP. Further studies are required to designate the exact causes behind the differences in CEI stability, in which focus must be 1) on the identification of gaseous and soluble side-products and 2) on the Fig. 2 . 2 Fig. 2. Surface chemistry of pristine, electrolyte soaked (OCV) and cycled graphite electrodes containing CMC (top) and PVdF-HFP (bottom). Electrodes were extracted after the 1st, 2nd, 10th and 125th cycles, at the discharged state (3.0 V vs Li + /Li). Survey spectra are provided to the left, followed by high-resolution C 1s, O 1s and F 1s spectra. The asterisk symbol (*) denotes species only encountered in the PVdF-HFP system.
Fig. 3 . 3 Fig. 3. In a), Raman spectra of pristine and cycled electrodes using CMC (top) and PVdF-HFP (bottom). Cycled electrodes were analysed at the discharged state, after the 1st, 2nd, 10th and 125th cycle. In b) and c), operando pressure measurements tracking gas evolution in the CMC and PVdF-HFP systems during the two first cycles.
|
10.3390/nano10061024
|
cc-by
| null | null |
openalex
|
Nanocrystalline diamond (NCD) field emitters have attracted significant interest for vacuum microelectronics applications. This work presents an approach to enhance the field electron emission (FEE) properties of NCD films by co-doping phosphorus (P) and nitrogen (N) using microwave plasma-enhanced chemical vapor deposition. While the methane (CH 4 ) and P concentrations are kept constant, the N 2 concentration is varied from 0.2% to 2% and supplemented by H 2 . The composition of the gas mixture is tracked in situ by optical emission spectroscopy. Scanning electron microscopy, atomic force microscopy (AFM), transmission electron microscopy, and Raman spectroscopy are used to provide evidence of the changes in crystal morphology, surface roughness, microstructure, and crystalline quality of the different NCD samples. The FEE results display that the 2% N 2 concentration sample had the best FEE properties, viz. the lowest turn-on field value of 14.3 V/μm and the highest current value of 2.7 μA at an applied field of 73.0 V/μm. Conductive AFM studies reveal that the 2% N 2 concentration NCD sample showed more emission sites, both from the diamond grains and the grain boundaries surrounding them. While phosphorus doping increased the electrical conductivity of the diamond grains, the incorporation of N 2 during growth facilitated the formation of nano-graphitic grain boundary phases that provide conducting pathways for the electrons, thereby improving the FEE properties for the 2% N 2 concentrated NCD films.Introduction Diamond is a versatile material whose properties make it interesting for a wide range of applications, from abrasives to power electronics . Among these applications, polycrystalline diamond-based field emitters have attracted significant interest in vacuum microelectronics due to their negative electron affinity for electron emission, strong bonding structure, extreme hardness to withstand ion bombardment, and good thermal and electrical conductivities to handle high currents [2, 3] . Chemical vapor deposition (CVD) is the most used technique for producing polycrystalline diamond. Using methane and hydrogen as gas precursors, it is possible to obtain diamond films with grain sizes ranging from tens of nanometers to hundreds of microns. Microcrystalline diamond (MCD) films possess micron-sized grains resembling monocrystalline diamond, implying a wide bandgap and hindering the field electron emission (FEE) behavior because of a shortage of electrons in the conduction band . As grain boundaries are known to be more conductive due to the presence of sp 2 -bonded carbon, efforts must be directed to a reduction of the diamond grain size. Nitrogen addition in CH 4 /H 2 plasma effectively reduces the size of the diamond grains from micron to nanosized, yielding nanocrystalline diamond (NCD) films. It also facilitates the formation of nanographitic phases in the grain boundaries, for which an improvement in the electrical conductivity and subsequent FEE properties have been demonstrated . The grain boundary conduction mechanism has been proposed for the enhancement of FEE properties of these N-doped NCD films [9, 10] . Still, the nanosized diamond grains are insulating in nature, which limits conduction to the grain boundaries, thereby limiting the FEE properties of N-doped NCD films. On the other hand, it is known that P forms a donor in both single as well as polycrystalline diamond, with the P atom mostly incorporating in substitutional positions in the grains [11, 12] . Here we present an approach to synthesize phosphorus and nitrogen co-doped NCD films, with the aim of improving the FEE properties of N-doped NCD films. With the target of obtaining a large proportion of conducting grain boundary phases (due to N-doping) between conducting P-doped diamond grains, P and N co-doped NCD films were deposited on Si substrates by microwave plasma-enhanced chemical vapor deposition (MWPECVD), and the optimum growth conditions to enhance the FEE properties were determined. The composition of the gas mixture during growth was tracked in situ by optical emission spectroscopy (OES). The resulting NCD films were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, transmission electron microscopy (TEM), and conductive AFM (C-AFM).
Materials and Methods A total of five 10 × 10 mm 2 n-type mirror-polished (100)-oriented silicon substrates (10-20 kΩcm) were exposed to an O 2 plasma for 1 min at 200 W and 50 sccm total flux in order to prepare the surface for diamond seeding. Oxygen plasma was used to clean the surface of organic contamination and to improve the hydrophilicity of the surface. Substrates were then seeded by drop-casting with a water-based colloidal suspension of ultra-dispersed detonation nanodiamond of size 6-7 nm (NanoCarbon Institute Co., Ltd., Japan.) and subsequent spin-drying . Growth was carried out in an MWPECVD ASTeX reactor. A mixture of CH 4 , H 2 , N 2 , and PH 3 gases was used with a total flux of 500 sccm. CH 4 and PH 3 were kept constant at 1% and 0.01%, respectively, in all the deposition experiments, while the proportion of N 2 was varied from 0.2% to 2% and complemented by H 2 . The working pressure and microwave power were set at 70 Torr and 2100 W, respectively, leading to a temperature of the growth surface in the range of 760 to 800 C, which was measured by a single-color optical pyrometer with the emissivity set to 0.3. An AvaSpec-2048 spectrometer with a spectral resolution of 1 nm per pixel was used to track the composition of the plasma region in contact with the sample by OES. All the samples were grown for a thickness of ~200 nm. Table 1 summarizes the growth conditions used for each sample. Grain morphology was studied by SEM using an FEI Quanta 200 FEG operated with 15 kV accelerating voltage, while surface roughness was evaluated by AFM using a Bruker Multimode 8 in tapping mode. The crystalline quality of the films was examined using a Horiba Jobin Yvon T64000 Raman spectrometer equipped with a BXFM Olympus 9/128 microscope, in combination with a Horiba Jobin Yvon Symphony CCD detector and a 488 nm Lexell SHG laser. The deposited NCD layers were studied in planar-view preparation by TEM at 200 keV accelerating voltage using a JEOL 2100 TEM. TEM samples were prepared by mechanical polishing and ion milling. Room-temperature field electron emission (FEE) measurements of the NCD films were carried out by a diode-type apparatus inside a vacuum chamber at a pressure of 2 × 10 -8 mbar. An Mo rod of 3 mm diameter acted as an anode, and the NCD films were used as the cathode. A micrometer feed-through with 1 μm resolution was used to control the anode-to-cathode distance. A Keithley 6517B electrometer (Keithley Instruments, Inc., Cleveland, OH, USA) was used to acquire the current-voltage (I-V) characteristics. C-AFM (NX-10, Park Systems) measurements were carried out at a negative substrate bias voltage of -5 V using an Au-coated silicon cantilever (NPG-10) from Bruker.
Results and Discussion SEM observations were carried out in order to analyze the changes in the grain morphology of the NCD films. The SEM micrograph of the 0.2% N 2 NCD sample (Figure 1a ) showed a larger grain size of 1 μm diameter for the biggest grains. Most of the grains looked (111)-faceted, with some not very evident re-nucleation in the edges. Figure 1b shows the surface of the sample growth at 0.35% N 2 , where the effect of the increase of nitrogen on the grain morphology is clear. Grains were drastically reduced to nanometric scale, where the largest ones were only a few hundred nanometers in diameter. Facets were not as evident in this case. The grain size continued reducing, and the shape became so-called cauliflower-like when nitrogen content was increased. The NCD sample grown at 2% N 2 , shown in Figure 1e , clearly exhibited small grains with many different facets in a clear cauliflower morphology that predicted larger grain boundary regions. Figure 2 shows AFM micrographs of each sample. The average roughness reduced with increasing nitrogen content, resulting in the Ra values of: 67, 40, 34, 32, and 30 nm for 0.2%, 0.35%, 0.5%, 1%, and 2% N 2 content, respectively. Thus, from 0.2% to 0.5%, there was a huge reduction of roughness, indicating a big change in the morphology of the diamond grains. The surface remained smoother at higher nitrogen concentration, but variations were not as abrupt. These results were in good agreement with the SEM micrographs (cf. Figure 1 ). Figure 3 shows the Raman spectra of these samples. This technique allowed the evaluation of the crystalline quality of the NCD layer by exploring the bonding character of the carbon phases present in the samples. The two peaks located at about ~1350 cm -1 (D-band) and ~1580 cm -1 (G-band) are characteristic of carbon materials, and correspond to breathing and stretching modes in sp 2 -bonded carbon clusters, respectively [14, 15] . Moreover, a third characteristic peak (labeled "dia") at ~1332 cm -1 was observed that relates to sp 3 diamond bonds. This peak looked sharp due to the microcrystalline character of the diamond film and ensured the diamond phase of the deposited film. The sharpness of the "dia" peak was related to defects and/or stress in the diamond phase and could be estimated by its FWHM. After Lorentz deconvolution, results of 14.0 ± 0.5, 15.7 ± 0.7, 12.8 ± 0.8, 20.3 ± 1.0, and 16.2 ± 1.4 cm -1 were obtained for 0.2%, 0.35%, 0.5%, 1%, and 2% nitrogen, respectively. There was no straightforward correlation between the FWHM and the nitrogen content. Consequently, there was no evidence of any effect on the stress or crystalline quality due to the addition of nitrogen to the gas mixture during growth. Nevertheless, this peak reveals that the diamond phase remained in all samples. A fourth peak appeared in the v band at ~1140 cm -1 , which is the specific mode corresponding to trans-polyacetylene (t-PA) segments and the vibration of sp 2 carbon atoms bonded at the grain boundaries . A second v band was at ~1460 cm -1 and overlapped the G band. Raman spectra showed the increase of the v band with N 2 concentration, which resulted in the formation of nanosized diamond grains (in concordance with SEM micrographs (cf. Figure 1 ) and, consequently, more sp 2 carbon bonded grain boundaries . The sp 2 /sp 3 ratio was estimated by the comparison of "dia", D, and G bands using the I dia /(Id + Ig) ratio. Figure 3b shows this ratio, after deconvolution, as a function of the nitrogen concentration. There was an increase of the sp 2 phases with the increase of nitrogen in the gas phase that tended to stabilize at 1%. The microstructure of the NCD samples was examined using TEM. Figure 4a shows a bright field (BF) TEM micrograph of the 0.2% N 2 NCD sample. The selected area electron diffraction (SAED) pattern of an area 1 μm in diameter is shown as an inset of Figure 4a . Bigger grains that were hundreds of nanometers in size are evidenced, one of which is shown in the dark field (DF) mode in Figure 4b . The sample grown with 2% nitrogen is shown in BF mode in Figure 5a . The smaller grains required higher magnification. SAED was performed in the area with an aperture of 200 nm diameter (Figure 5b ). Three different reflections in DF mode are provided in Figure 5c -e to show the grains that were tens of nanometers in size. Figure 6a shows the FEE current density (J) versus applied field (E) of all co-doped NCD samples. The FEE results were Fowler-Nordheim (FN) modeled , from which the turn-on field (E 0 ) was determined for each film, as shown in Figure 6b . These values, summarized in Table 2 , show a drastic reduction of E 0 for higher N 2 content combined with a big increase in the current density. In this way, for the 0.2% N 2 sample, the turn-on field was 83.7 V/μm, and the current density at 53 V/μm applied field was 10 -5 mA/cm 2 , respectively. Similar values were obtained for 0.35% N 2 (E 0 = 88.5 V/μm and J = 10 -5 mA/cm 2 ) even if the SEM micrographs showed clear differences in the grain morphology between the 0.2% N 2 and 0.35% N 2 NCD samples (cf. Figure 1 ). Nevertheless, at an N 2 ratio of 0.5%, the electric field for which the film can be turned on was reduced to half (i.e., E 0 = 41.0 V/μm), while the current density increased to J = 22 × 10 -5 mA/cm 2 . Interestingly, for the 2% N 2 sample, E 0 reached its minimum value of 18.7 V/μm, with the current density reaching 13 × 10 -2 mA/cm 2 . Moreover, the β values were estimated from the slope (m) of FN plots (see the arrow in Figure 6b ) using the relation β = [-6.8 × 10 3 φ 3/2 ]/m where φ is the work function, and m is the slope of the FN plot. The calculated β values were 27, 12, 130, 89, and 230 for 0.2%, 0.35%, 0.5%, 1%, and 2% N 2 content, by taking the φ value as 5.0 eV, respectively . The nanosized diamond grains with wider grain boundaries in the 2% N 2 NCD sample were the main factors for the larger β value. Consequently, the 2% N 2 sample showed better FEE characteristics, viz. lower E 0 value, higher J value, and larger β values as compared with the other co-doped samples in this study. To determine the reason for the enhancement of the FEE characteristics of the 2% N 2 NCD sample, C-AFM measurements were carried out to detect the current conduction paths of the NCD films. Figure 7a ,c shows the C-AFM topographic image and the corresponding current signal for 2% N 2 NCD. C-AFM measurements of the 0.2% N 2 NCD sample were also carried out for comparison (Figure 7b, d ). The brighter contrast in the current images signifies a larger emission current. For both samples, larger emission current was observed from the grain boundaries, which was due to the presence of sp 2 -bonded carbon phases at the grain boundaries confirmed from the Raman results (Figure 3 ). Harniman et al. also observed that FEE from diamond surfaces originates preferentially from the grain boundaries and not from other topographical features. This was observed in low-doping samples where grain boundaries remained relatively conductive compared to the bulk grains. In these samples, the electrons used the grain boundaries as a path to climb from the base of the film to the surface, where they were emitted. The situation changed when the grains were conductive, causing some of the current to be transported through the grains themselves and be emitted from the facets. In our results, the number density of highly conductive regions was larger for 2% N 2 NCD than that of 0.2% N 2 NCD because 2% N 2 NCD films contained more sp 2 -bonded phases at the grain boundaries, which was in accordance with the Raman studies. Moreover, emission sites were also observed from the P-doped diamond grains of 2% N 2 NCD. Hence the C-AFM measurements clearly illustrate the existence of a high density of local electron emission sites in 2% N 2 NCD, implying the formation of conductive nanochannels at the grains and grain boundaries resulting in the enhanced FEE properties . In our previous studies, it was observed that phosphorus was doped directly inside the lattice of the diamond , which made the diamond grains conductive. However, the main factor responsible for the reduction of nanosized diamond grains and the induction of sp 2 -bonded carbon phases in the grain boundaries is still not clear. To determine the factors responsible, we carried out in situ OES measurements during the sample growth. Figure 8a shows optical emission spectra taken from the plasma during NCD sample growth from CH 4 /H 2 /N 2 /PH 3 gas mixtures with varying N 2 concentrations. The H α and H β lines are the Balmer atomic hydrogen emission lines observed at ~655 and ~484 nm [20, 21] , respectively, whereas the CN violet system (386.3 and 418.1 nm) and the N 2 line at 357.3 nm were induced due to the addition of N 2 . The intensity of CN and N 2 lines increased as the N 2 concentration in the plasma increased, and reached a maximum for 2% N 2 NCD (Figure 8 , spectrum V). Generally, the CH 4 species in the plasma dissociated and formed CH x species (x = 1, 2, 3). However, the CH lines were overlapped by the CN line [23, 24] , and lines at 386.3 and 418.1 nm were marked as CN + CH. The C 2 swan system at 516.0 nm was also observed with low intensity . Our previous studies stated that the presence of CH and C 2 species are responsible for the origin of nanosized diamond grains because the C 2 species re-nucleate diamond easily, whereas the CH species passivate the diamond nuclei and efficiently result in the formation of nanosized diamond grains [10, 25] . Based on the recorded spectra, Figure 8b shows a comparative graph of the species in the plasma during the growth. At a high concentration of N 2 (2%) in the plasma, the CN + CH peaks increased. This was probably due to an increase of the CN species that were more present as compared to the CH species . Moreover, the NCD films were grown at a high growth temperature (>700 C), which made it energetically favorable for the CN species to be more active, likely resulting in the formation of sp 2 -bonded carbon at the grain boundaries [10, 26] . Hence CN, CH, and C 2 species were the main factors for the origin of the nanosized diamond grains and the induction of sp 2 -bonded carbon phases in the grain boundaries of 2% N 2 NCD films.
Conclusions From this study, the combined effect of PH 3 and N 2 on the drastic reduction of diamond grain size and the consequent enlarging of the grain boundaries was clearly evidenced. Roughness reduction was illustrated by AFM, while the reduction of grain size was pointed out by SEM and TEM. C-AFM studies showed that more emission sites from diamond grains and sp 2 -bonded carbon grain boundaries of an NCD sample co-doped with P and N, with 2% N 2 used in the gas phase, resulting in enhanced FEE properties, viz. a low E 0 value of 18.7 V/μm, a high current density of 13 × 10 -2 mA/cm 2 , and a field enhancement factor of 230. The CN, CH, and C 2 species in the growth plasma were considered the prime factors for the origin of the nanosized diamond grains and the induction of sp 2 -bonded carbon phases in the grain boundaries of the co-doped NCD films. Figure 1 . 1 Figure 1. SEM micrographs of the samples grown with (a) 0.2% N 2 , (b) 0.35% N 2 , (c) 0.5% N 2 , (d) 1% N 2 and (e) 2% N 2 .
Figure 2 . 2 Figure 2. Topographic AFM images of the samples grown with (a) 0.2% N 2 , (b) 0.35% N 2 , (c) 0.5% N 2 , (d) 1% N 2 and (e) 2% N 2 .
Figure 3 . 3 Figure 3. (a) Raman spectra of the NCD samples grown with different N 2 concentrations. (b) Dia and G + D bands ratio (I dia /(Id + Ig)) for each nitrogen content.
Figure 4 . 4 Figure 4. (a) Bright field (BF) TEM micrograph of sample grown with 0.2% N 2 . The inset of (a) shows the SAED pattern using an aperture with 1 μm diameter. (b) Dark field (DF) micrograph of one reflection that shows one of the grains with good contrast.
Figure 5 . 5 Figure 5. (a) BF TEM micrograph of the sample grown with 2% N 2 . (b) SAED pattern of the region shown in (a) using an aperture with 200 nm diameter. (c-e) DF micrographs of the reflections marked on the SAED pattern.
Figure 6 . 6 Figure 6. (a) FEE current density (J) as a function of applied field (E). (b) Fowler-Nordheim (FN) plots of the corresponding J-E characteristic curves. All the results are shown for the five samples grown at different N 2 contents.
Figure 7 . 7 Figure 7. C-AFM topographic images (a,b) and the corresponding current signal (c,d) of 2% N 2 NCD and 0.2% N 2 NCD samples, respectively.
Figure 8 . 8 Figure 8. (a) OES spectra recorded during the deposition process of the five samples labelled by their N 2 amount. (b) Intensity of C 2 , N 2 , and CN + CH peaks normalized to the H α peak for each nitrogen content used.
Table 1 . 1 Growth conditions used during the microwave plasma-enhanced chemical vapor deposition (MWPECVD) deposition of the nanocrystalline diamond (NCD) layers, Ra determined by AFM and full width at half maximum (FWHM) of the dia peak, corresponding to the Raman spectrum. Power Pressure Temperature CH 4 PH 3 N 2 Ra FWHM "dia" 2100 W 70 Torr 760-800 C 1% 250 sccm 0.2% 67 nm 14.0 cm -1 2100 W 70 Torr 760-800 C 1% 250 sccm 0.35% 40 nm 15.7 cm -1 2100 W 70 Torr 760-800 C 1% 250 sccm 0.5% 34 nm 12.8 cm -1 2100 W 70 Torr 760-800 C 1% 250 sccm 1% 32 nm 20.3 cm -1 2100 W 70 Torr 760-800 C 1% 250 sccm 2% 30 nm 16.2 cm -1
Table 2 . 2 Field electron emission (FEE) properties of P and N co-doped NCD films with varying N 2 concentrations. N 2 E 0 (V/μm) β J at 53 V/μm 0.2% 83.7 27 1 × 10 -5 (mA/cm 2 ) 0.35% 88.5 12 1 × 10 -5 (mA/cm 2 ) 0.5% 41.0 130 22 × 10-5 (mA/cm 2 ) 1% 36.2 89 26 × 10 -4 (mA/cm 2 ) 2% 18.7 230 13 × 10 -2 (mA/cm 2 )
|
10.1186/s13048-019-0498-0
|
cc-by
| null | null |
openalex
|
Background: Increased activity of the chaperones Hsp70 and Hsp90 is a common feature of solid tumours. Translocase of the outer mitochondrial membrane 34 (Tomm34) is a cochaperone of both Hsp70 and Hsp90 that was found to be overexpressed in colorectal, hepatocellular, lung and breast carcinomas. The expression profile of Tomm34 in ovarian cancer has not been investigated. Therefore, the aim of the current study was to investigate the expression pattern of Tomm34 in ovarian carcinomas and analyse its correlation with clinico-pathological parameters. Results: Epithelial ovarian cancers (140) were histologically classified based on their morphology and graded into two types comprising 5 histologic subgroups. Type I carcinomas comprise low grade serous (LGSC), clear cell (CCOC) and endometrioid (ENOC), type II comprises high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). Tomm34 was more highly expressed in type II than type I carcinomas (p < 0.0001). Comparing tumours based on the mutation in the TP53 gene revealed similar results, where mutant tumours exhibited significantly higher levels of Tomm34 (p < 0.0001). The decreased levels of Tomm34 in type I carcinomas were particularly evident in clear cell and mucinous carcinomas. The expression of Tomm34 was also positively correlated with FIGO stage (r = 0.23; p = 0.007). Tomm34 levels also indicated poor prognosis for patients with mutant p53.
Conclusions: Our data indicate that Tomm34 is commonly expressed at high levels in epithelial ovarian cancers, except for the clear cell and mucinous subtypes. The expression of Tomm34 corresponds with the dualistic model of ovarian cancer pathogenesis where high grade, type II tumours exhibit higher expression of Tomm34 in contrast to type I tumours. These data are also comparable to the previous findings that Tomm34 is a marker of progression and poor prognosis in human cancer.Background Epithelial ovarian cancer accounts for approximately 90% of ovarian tumours and is usually diagnosed only at advanced stages of the International Federation of Gynecology and Obstetrics (FIGO) system, accounting for its high mortality rate [1, 2] . Epithelial ovarian cancer is not a single disease and a dualistic model has been proposed to describe morphological subtypes and cell of origin [2, 3] . Type I ovarian cancers are low grade and develop slowly (including endometrioid, clear cell, mucinous and low grade serous adenocarcinomas) whereas type II cancers are the most common form, representing highgrade serous adenocarcinomas. It is also recognized that high and low-grade serous adenocarcinomas originate from precursor lesions in the fallopian tube, whilst the other histological type I tumours arise from endometriosis, germ cells or transitional cells, with an important role for distinct genetic alterations influencing the tumour characteristics . Most notably, mutations in the p53 tumour suppressor are an overwhelming characteristic of Type II high grade serous tumours [7, 8] . Whilst many low grade lesions can be treated with surgery alone, high grade epithelial ovarian cancer is a difficult-to-treat disease that requires surgery plus combination chemotherapy, and even then recurrence is common (70-80% within two years), although this is improving with current targeted therapies for subsets of patients . Increased chaperone activities are a universal feature of cancer and anti-Hsp90 or anti-Hsp70 therapeutics are under investigation for the treatment of various cancer types , including ovarian cancer [11, 12] . Chaperone activities are dependent on their interactions with cochaperones that provide either protein folding or protein degradation functions. The protein folding activity of chaperones is hyperactive in cancers due to enhanced interactions of phosphorylated Hsp90 with Hsp70/Hsp90-organising protein (HOP, also known as STIP1) and reduced interaction with C-terminal Hsp70 interacting protein (CHIP, also known as STUB1) . Translocase of the outer mitochondrial membrane 34 (Tomm34) is an additional component of the cellular chaperone system involved in protein folding. As the name suggests, Tomm34 was initially identified as being involved in mitochondrial protein processing [14, 15] . Subsequent studies have shown that Tomm34 interacts with both Hsp70 and Hsp90 and modifies their protein folding activities . In cancer, high levels of Tomm34 have been reported in bladder, colorectal and breast cancers compared to their normal tissue counterparts . In these cancers, Tomm34 promotes colorectal cancer cell growth and is a biomarker of poor outcome in early invasive breast cancer and bladder cancer . As a tumour-associated protein, Tomm34 peptide vaccination is under investigation as a therapeutic option for colorectal cancer, with significant Tomm34 cytotoxic T-lymphocyte (CTL) response observed . Previous data have indicated that the ovary also expresses TOMM34 mRNA although its expression in ovarian cancer has not been reported. Here, we investigated the levels of Tomm34 in ovarian cancers of mixed subtypes using immunohistochemistry. The data were correlated with tumour type and clinicopathological variables and demonstrate that Tomm34 is expressed at high levels in type II carcinomas and correlates with high FIGO stage.
Results
Patient details The patients (136) ranged in age from 29 to 86 years old (mean 59, median 59). Histological diagnosis classified tumours based on their morphology and grade into two types comprising 6 histologic subgroups. Type I carcinomas comprise low grade serous (LGSC), clear cell ovarian carcinoma (CCOC), endometrioid ovarian carcinoma (ENOC) and mucinous ovarian carcinoma (MOC). Type II include high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). Since the carcinomas with serous and endometrioid morphology exhibited higher heterogeneity, the assignment of individual samples to histological subtypes was further verified by analysis of p53 status using sequencing and immunohistochemistry (Fig. 1 ). Serum CA125 levels varied from 6.6-42,415 U/ml, with 6 patients showing levels of less than 35. Thirty four (25%) cancers were FIGO stage 1; 15 (11%) FIGO 2; 65 (48%) FIGO 3 and 20 (15%) FIGO 4 (1 missing, n = 135). Fifty-one patients (38%) had residual disease (3 missing, n = 133) and 46 patients were alive and 90 were dead at last follow up. Twenty patients (14%) had a secondary tumour. Detailed information on individual cases is given in additional file 1.
Tomm34 staining Tomm34 staining was seen in the cytoplasm of tumour cells. In the cohort of 136 ovarian cancers, Tomm34 was absent (score of 0) in 14 tumours, 35 cancers were scored as class 1; 44 as class 2; and 43 as class 3 (Fig. 2 ). We tested whether the level of Tomm34 corresponds with the histological type of tumour and whether it correlates with the dualistic model dividing the tumours into 2 types according to their pathogenesis. Figure 3a shows lower expression levels of Tomm34 in type I tumours (MOC, CCOC, LGSC and ENOC) compared to type II tumours (SET and HGSC). The lowest levels of Tomm34 were detected in MOC and CCOC (10 and 11 cases) when compared to the other samples t(138) = 5.6; p < 0.0001. The most statistically significant differences in Tomm34 were found when the cohort was classified according to tumours of types I and II t(138) = 6.6; p < 0.0001 (Fig. 3b ). Comparing tumours bearing mutation in the TP53 gene (94 cases) with TP53 wild-type tumours (39 cases; 7 missing) revealed that TP53 mutant tumours exhibited significantly higher levels of Tomm34 t(130) = 4.7; p < 0.0001. Tomm34 staining also correlated with tumour progression represented by FIGO scale (Fig. 4 ). In accordance with this finding, higher values of Tomm34 were also found in patients who had residual disease after surgery (n = 51) versus the rest (n = 82) using independent samples t-test (p = 0.036). We also found that high Tomm34 levels correlate with pT status but not with presence of secondary tumour or response to therapy (Table 1 ). Analysis of patients with serous cancers as a separate group showed that Tomm34 staining correlated with pT status and high FIGO stage, but not with tumour grade or any of the other variables analysed (Table 2 ). To investigate the effects of Tomm34 on patient survival in more detail, we also analysed publicly available data using Kaplan-Meier Plotter for Ovarian Cancer (http://kmplot.com/analysis/index.php?p=ser vice&cancer=ovar). These analyses indicated that Tomm34 mRNA levels associate with poor overall survival (p = 0.036; HR = 1.
Discussion Cancer cells require a high level of protein synthesis due to their rapid proliferation and exhibit a high degree of stress due to genomic instability and lack of both oxygen and essential nutrients. To overcome such difficulties, cancer cells show up-regulation of many stress-induced proteins, including components of the protein chaperone system, particularly Hsp70 and Hsp90 . Chaperone inhibition is therefore a clinically promising area , including for the treatment of ovarian cancer [11, 12] . Metabolic abnormalities are also a common finding in human cancers and the high rate of glucose uptake by tumour cells is utilised clinically for cancer imaging by 18F-2-deoxyglucose uptake and accumulation. Although it was originally thought that aerobic glycolysis in cancer cells (the Warburg effect) represented a lack of mitochondrial function, it is now clear that altered mitochondrial function represents a redeployment of glycolytic nutrients from catabolism to anabolism, required to meet the biosynthetic requirements of rapidly proliferating cancer cells, and NADPH production to help maintain redox balance . These alterations to mitochondrial function relate to altered chaperoning of mitochondrial proteins . Thus, targeting abnormal mitochondrial function and chaperoning of mitochondrial proteins is also a promising strategy for cancer therapeutics . Hsp90 and Hsp70 play critical roles in mitochondrial protein stabilisation and folding and various co-chaperones such as p23, Hsp40 and HOP are also involved . In this respect, Tomm34 was originally identified as a potential mitochondrial import protein and Tomm34 antibodies inhibit transport of preproteins into the mitochondria, whilst expression stimulates mitochondrial preprotein maturation and TOMM34 siRNA inhibits this process . Tomm34 is also reported to be increased as a component of compensatory adaptations to maintain normal rates of protein import in response to mitochondrial abnormalities . On the other hand, and in agreement with our immunostaining data, Tomm34 exists predominantly in the cytoplasm rather than in mitochondria, suggesting it is involved in the transport of mitochondrial preproteins in an unfolded state prior to import [14, 17, 44] . Our data indicate that Tomm34 is commonly expressed at high levels in human ovarian cancers, except for the MOC and CCOC subtype, where high level Tomm34 is rarely seen. Within the different sub-types of ovarian cancer, high levels of Tomm34 associate with higher stage and higher grade cancers and similar findings are seen within the sub-type of serous cancers. Most notably, we have found that Tomm34 associates with poor survival of patients with p53-mutant ovarian cancers. These data are also comparable to the findings that Tomm34 is a marker of poor outcome and a predictor of distant metastasis in breast cancer [22, 23] . Thus, as with breast cancer patients, A Fig. 3 Expression of Tomm34 according to histological classification. a Distribution according to histological group. b The independent samples t-test revealed the most significant difference in Tomm34 between type I and type II tumours Tomm34 may serve as part of a panel of markers for prognostic determination in ovarian cancers. In this respect, proteomic analysis of an animal model of ovarian cancer revealed up-regulation of numerous proteins involved in metabolic processes, including endoplasmic stress responses, mitochondrial systems and chaperones such as Hsp70 and Grp78 . The mechanism for high level expression of Tomm34 is unclear from our studies. Data from the COSMIC database (http://cancer.sanger.ac.uk/cosmic) indicate that only 1.5% of 729 ovarian cancers tested show copy number variation gain of TOMM34, indicating that gene amplification is a rare event. However, Tomm34 is transcriptionally regulated by NRF-1 and NRF-2 [46, 47] and these latter are implicated in directing metabolic reprogramming during stress and are often hyperactive in cancers. Although further research will be required to elucidate the mechanisms that regulate Tomm34 in ovarian and other cancers, our data imply that Tomm34 has pro-tumourigenic actions and may serve as a useful prognostic indicator and potential therapeutic target in ovarian cancers.
Conclusions The co-chaperone Tomm34 is frequently expressed in epithelial ovarian cancers. Immunohistochemical identification of Tomm34 may be a useful adjunct to provide prognostic information and may serve as an immunogenic target for therapy.
Methods Ovarian cancer samples and TMA construction A total of 140 samples of ovarian cancer with anonymised clinicopathological and survival data were available for immunohistochemical staining of Tomm34. All tissues had been removed during surgery, fixed in formalin and processed into paraffin wax for histopathological diagnosis. Tissue microarrays were constructed using five separate cores (1.5 mm diameter each) selected from different regions of each tumour. Clinicopathological information includes age, histological subtype, p53 status, TNM status, grade, FIGO stage, CA125 levels, tumour response to therapy, progression free survival and overall survival. Histological classification was performed as follows: type I carcinomas comprise low grade serous carcinoma (LGSC), clear cell ovarian carcinoma (CCOC), endometrioid ovarian carcinoma (ENOC) and mucinous ovarian carcinoma (MOC). Type II include high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). The study was approved by the MMCI biobank and all patients gave informed consent for the use of their tissues for research.
Tomm34 antibody generation and characterization Full-length recombinant Tomm34 purified from E. coli was used for immunization of mice. The hybridomas producing monoclonal antibodies were generated by Moravian Biotechnology. The clones were characterized by immunoblotting and immunohistochemistry. Hybridoma clone Tomm34-4.1 was used due to its highest sensitivity and specificity in immunohistochemistry. Specificity of the antibody was tested on MCF7 Tomm34 -/cell line, where the Tomm34 gene was removed by CRISPR knock-out (Additional file 3).
microarrays, immunohistochemistry and scoring Tissue blocks of primary tumours were fixed in 4% neutral formaldehyde for approximately 24 h before processing into paraffin wax. Representative cores of tumour were selected from the diagnostic blocks by an experienced histopathologist. The tissue microarrays comprised 45 tissue cores consisting of nine tumour samples each represented by five cores from the original paraffin block. Endogenous peroxidase was blocked with 3% hydrogen peroxide in phosphate buffered saline (PBS), pH 7.5, for 15 min. Antigen retrieval was performed in 1 mM EDTA-NaOH (pH 8.0) for 40 min at 93 C. Primary antibodies were applied overnight at 4 C at 2 μg/ml in antibody diluent (DakoCytomation, Denmark). The antibody was visualized using peroxidase labelled polymer conjugated to goat anti-mouse immunoglobulins and EnVision+ System containing DAB chromogen (DakoCytomation, Denmark). The nuclei were counterstained with Gill's haematoxylin before permanent mounting. Neoplastic cells were classified into four staining intensities: 0 no staining, 1 weak staining, 2 moderate staining, 3 strong staining.
Statistical analyses The independent samples t-test was used to compare two sets of independent of and identically distributed samples; P values < 0.05 were considered significant. Relationships between two continuous variables were tested by Pearson correlation where correlation coefficient r was considered significant if P values < 0.05. Fig. 1 1 Fig. 1 Distribution of histologic groups of 140 epithelial ovarian cancers. High grade tumours with endometrioid morphology and TP53 mutation were classified as type II SET subgroup tumours
29 (1.02-1.64; n = 506), poor progression-free survival (p = 0.0026; HR = 1.42 (1.13-1.8); n = 483) and poor post-progression free survival (p = 0.012; HR = 1.4 (1.07-1.82); n = 325) in patients with mutant p53 cancers (Additional file 2).
Fig. 2 2 Fig. 2 Representative staining patterns of Tomm34 in ovarian cancer. a histoscore 0, CCOC, grade 3. b histoscore 1, ENOC, grade 1. c histoscore 2, HGSC, grade 3. d histoscore 3, HGSC, grade 3. Magnification 100x
Fig. Fig. Relationship between FIGO stage and Tomm34 histoscore (0-3). Pearson correlation r = 0.23; p = 0.0066
Table 1 1 Tomm34 correlates with tumour grade, FIGO stage and tumour type in ovarian cancer (n = 140) Variable Tomm34 0-1 Tomm34 2-3 P value Tomm 34 score 50 90 pT 1-2 2 9 2 2 pT 3-4 19 57 0.0004 pN 0 19 18 pN 1 11 16 0.454 M0 44 59 M1 4 15 0.123 Secondary No 30 76 Secondary Yes 8 11 0.456 Refractory 2 4 Resistant 7 15 Sensitive 29 63 0.966 pT size of primary tumour, pN degree of spread to regional lymph nodes given by histopathologic examination and M presence of distant metastasis. Tomm34 0-1 = IHC score of 0 or 1; Tomm34 2-3 = IHC score of 2 or 3; P value = Fisher exact 2-tailed test
Table 2 2 Tomm34 staining in serous ovarian cancers (n = 102) Variable Tomm 0-1 Tomm 2-3 P value Tomm 34 29 73 pT 1-2 1 5 1 6 pT 3-4 12 49 0.0071 pN 0 8 13 pN 1 7 14 1 M0 26 48 M1 2 14 0.134 Secondary No 24 59 Secondary Yes 4 11 1 Refractory 0 4 Resistant 5 14 Sensitive 19 48 0.707 pT size of primary tumour, pN degree of spread to regional lymph nodes given by histopathologic examination and M presence of distant metastasis. Tomm34 0-1 = IHC score of 0 or 1; Tomm34 2-3 = IHC score of 2 or 3; P value = Fisher exact 2-tailed test
|
10.22541/au.167226820.06612648/v1
|
cc-by
| null | null |
openalex
|
When there are obstacles around the target point, the mobile robot cannot reach the target using traditional Artificial Potential Field (APF). Besides, the traditional APF is prone to local oscillation in complex terrain such as three-point collinear or semiclosed obstacles. Aiming at solving the defects of traditional APF, a novel improved APF algorithm named back virtual obstacle setting strategy-APF (BVO-APF) has been proposed in this paper. There are two main advantages of the proposed method. Firstly, by redefining the gravitational function as logarithmic function, the proposed method can make the mobile robot reach the target point when there are obstacles around the target. Secondly, the proposed method can avoid falling into local oscillation for both three-point collinear and semi-closed obstacles. Compare with APF and other improved APF, the feasibility of the algorithm is proved through software simulation and practical application.Introduction With the rapid development of artificial intelligence, mobile robots are widely used in various fields such as medical, industrial and military. For mobile robots, the path planning algorithm is equivalent to the eyes of the robot and plays a crucial role. How the robot chooses a safe and collision-free optimal path from the initial point to the target point is the research focus of the path planning algorithm. Commonly used algorithms are A* algorithm, Ant Colony Optimization, ACO, Genetic Algorithm, GA, Dijkstra algorithm , Rapidly-Exploring Random Tree, RRT, Artificial Potential Field, APF , etc. The A* algorithm introduces a heuristic function to avoid a large number of invalid search paths in the path planning problem. ACO is a heuristic random search algorithm, which has problems such as large amount of calculation and slow convergence. The main characteristics of the Dijkstra algorithm are the center spreads outward until it reaches the end point, but it traverses many computing nodes, and the efficiency is low. The path obtained by GA meets the actual path selection requirements, but the calculation amount is large and the time consuming is big, which cannot meet the real-time requirements. The main feature of RRT is that it generates a new node in space and continues to extend to the target point on the new node, but the generated path is not smooth enough, and the searched path is constantly changing. Bio-inspired algorithms , can yield good solutions, but they are not necessarily the best solution. All the mentioned algorithms have their respective advantages and disadvantages. In the APF method, you only need to know the current position of the mobile robot, the surrounding obstacles and the position of the target to be reached, and then you can know the moving position of the next part of the robot. It has the advantages of fast speed and smooth generated road strength, so the algorithm is widely used in the path planning of mobile robots. However, the APF method also has its own shortcomings. Firstly, it is easy to fall into local oscillation in complex terrain. Secondly, the robot often fails to reach the target point when there are obstacles near the target point. When the target point is far away, it is easy to collide with obstacles due to the excessive gravitational force generated. By adding a force based on the tangent line between the robot and the obstacle, Wang et al enabled the robot to escape when trapped in a local oscillation. But in the face of semi-closed obstacles, the robot cannot escape the local oscillation. Wang and Lee D et al set a virtual target point to make the robot escape from the local oscillation point by setting a virtual target point to provide gravity when the robot falls into local oscillation. Rostami added adjustment factors to the traditional artificial potential field function to bypass obstacles to overcome local minima and target unreachable problems. However, in the face of special semi-closed obstacles, there is no way to set virtual target points. He et al redefined the repulsion model of artificial potential field method by simulating the fading algorithm, and designed a controller that can escape local oscillation. The global path is smooth, but it still cannot be well solved in the face of complex path conditions. Xu et al proposed an improved artificial potential field method. Firstly, the concept of safe distance was proposed to avoid unnecessary paths, so as to solve the problems of long path length and long algorithm running time. Then, in order to avoid the robot being trapped in the local minimum and trap area, the predictive distance was introduced into the algorithm, so that the algorithm was able to react before the robot being trapped in the local minimum or trap area. Finally, the robot was guided to avoid the local minimum and trap area by setting the virtual target points reasonably. Chen and Huang described the inherent problems of artificial potential field and analyzes their causes. Then, aiming at the problem of local minimum trap, the virtual obstacle method is proposed to repel mobile robot from traps. Next, in order to solve the chattering phenomenon, a geometric method is proposed to ensure that the route is relatively smooth. Finally, computer simulations are carried out to demonstrate the effec-tiveness of these two methods. Neurofuzzy systems are also the algorithms that have served as the core methodology to develop path planning. It simulated the workspace of the robot as a network of neurons. Fan and Guo proposed the regular hexagon bootstrap method to improve the local minima problem. At the same time, a relative velocity method for moving object detection and avoidance is proposed for dynamic environment. However, when the obstacle suddenly accelerates or changes its direction of motion, it may cause collision. Montiel et al introduced a parallel evolutionary APF for the dynamic environment. It can achieve controllability in complex real-world sceneries with dynamic obstacles. Cheng et al proposed a novel integrated AUV path planning algorithm by combining the velocity synthesis and APF. Zheng and Edwin proposed a new algorithm based on an artificial potential field and hierarchical cell decomposition technique is developed to solve the find-path problem for a mobile robot. Liu and Zhao proposed an improved particle swarm optimization (IPSO) algorithm, which combines the PSO with the Zigzag algorithm to preplan the search routes and improve the efficiency of the UAVs to search the plume. The improved artificial potential field method is used to avoid dynamic obstacles effectively,and the velocity synthesis algorithm is used to achieve the optimal path. In view of the problems and deficiencies mentioned above, a novel improved APF algorithm named BVO-APF has been proposed. The main contributions of this paper are concluded as follows. (1).By redefining the gravitational formula as logarithmic function of traditional APF, the proposed BVO-APF algorithm can reach the target point when there exist obstacles around it. When there are obstacles around the target point, the gravity received by the robot is too small to overcome the repulsion, the robot cannot reach the target point. To solve the above problems, we redefine the gravitational formula of APF. According to the definition of logarithmic function proposed in this paper. When the robot approaches the target point, the gravity obtained by the robot will overcome the repulsion. Eventually the robot will reach the target point. (2).In the BVO-APF algorithm, tangent algorithm and back virtual obstacle setting strategy are proposed. The robot can avoid falling into local oscillation for both three-point collinear and semi-closed obstacles. (3).In this paper, the algorithm proposed in this paper is simulated and contrasted through the Matlab software simulation platform and the ROS vehicle simulation platform. The algorithm proposed in this paper has good feasibility and practicability, no matter in simulation experiment or entity demonstration.
Basic principle of artificial potential field method The APF was first proposed by Khatib as a virtual force method. His core idea is to put the robot in the surrounding environment and design it to move in an abstract artificial gravitational field. There are two types, one is the gravitational force between the robot and the target point, and the other is the repulsive force between the robot and the obstacle. The resultant force provides the driving force for the robot. The gravitational force of the robot on the obstacle is U att , the coordinate of the target point is (x o , y o ),the position of the real-time center point of the robot is (x, y). The APF defines the gravitational function as : U att = 1 2 ερ 2 o , ρ o = (x + x o ) 2 + (y + y o ) 2 , where ρ o is the Euclidean length from the center coordinates of the robot to the coordinates of the target point, ε is the gain coefficient of the gravitational potential field. With the gravitational function, the gravitational force of the APF is the derivative of the gravitational field to the distance. F att = -U att = ερ o , where F att represents the gravitational force on the robot. The repulsive field formula U rep F rep are defined as: U rep = 1 2 μ( 1 ρ ob -1 ρ ) 2 if ρ ob <= ρ 0 if ρ ob >= ρ (1) F rep = μ( 1 ρ ob -1 ρ ) 1 ρ 2 ob if ρ ob <= ρ 0 if ρ ob >= ρ (2) where ρ ob = (x -x ob ) 2 + (y -y ob ) 2 represents the distance between the robot and the obstacle. where ρ represents a safe distance between the robot and the obstacle. Outside this distance, the repulsion of the obstacle has no effect on the robot. When it is less than this threshold, the repulsion of the obstacle to the robot is affected by the distance between the two. When the robot is within the influence range of multiple obstacles, its repulsive force is the sum of multiple repulsive forces ,the resultant force of the repulsive force on the robot is : F rep = n i=0 F repi . So the formula for the combined force is: F joint = F att + n i=0 F repi (3) Although the APF is simple in structure and convenient for real-time control, it has some shortcomings. (1).Since the gravity function is inversely proportional to the distance between the robot and the obstacle, the farther the robot is from the target point, the greater the gravity it receives. When the robot moves away from the target point, the gravity exerted on the robot will be too high, causing the robot to hit the obstacle. (2).Due to the limitations of the APF, it is prone to local oscillation in more complex situations. The typical situation is three points and one line of robot,as shown in Fig 2 . When the repulsive force and gravity are equal and the direction is opposite, the robot will fall into local oscillation and cannot escape. In addition, when the robot is trapped in a semi-closed obstacle, the APF is also unable to jump out of local oscillation. (3).When the obstacle is very close to the target point, the target point may not be reached.
Back virtual obstacle setting strategy for APF In view of the shortcomings of APF, The algorithm redefines the traditional gravity formula, makes its path smoother, and solves the unreachable problem of the target point. In addition, for the general three-point collinear problem, this paper proposes the tangent algorithm, which can make the robot jump out of the local oscillation smoothly and reach the target point. Then for complex obstacles or even semi-closed obstacles, some algorithms can not solve this kind of problem well. The backward strategy this paper proposed is that when the robot enters the semi-closed obstacle and falls into local oscillation, it can return to the previous step of entering the semi-closedo bstacle. Then the semi-closed obstacles are completely enclosed by adding virtual obstacles, the robot avoids entering semi-closed obstacles again.
Definition of Gravitational field function of BVO-APF For the previous gravitational formula, this paper redefines the new gravitational function F att(q) . The gravitational function formula is as follows: F att(q) = n log 2 ( 1 ρ (q,qo ) +1 ) ifρ (q,qo) <= ρ 1 m log k (ρ (q,qo) + 1) ifρ (q,qo) >= ρ 1 (4) where ρ (q,qo) is the distance between the robot and the target, and its mathematical According to the definition of the gravitational field formula, when the robot is outside the limited distance ρ 1 the gravitational force is a single increasing function, but due to the nature of the logarithmic function, the force growth of the gravitational field is relatively smooth, and the problem of collision with obstacles due to excessive gravitational force will not occur. expression is ρ (q,qo) = (x -x o ) 2 + (y -y o ) 2 , (x, When the robot enters the limited distance ρ 1 , according to the formula shown in formula 4. At this time, if there are obstacles around the target point, the gravitational force overcomes the repulsive force when the robot approaches the target. In this way, the robot will eventually reach the target point.
Definition of Repulsive field function of BVO-APF The potential field of the repulsion is U 1 rep (x), the expression formula of U rep is the same as the traditional potential field method: U 1 rep (x) = 1 2 μ( 1 ρ ob -1 ρ ) 2 if ρ ob <= ρ 0, if ρ ob >= ρ (5) where ρ ob represents the distance between the robot and the obstacle, its mathematical formula is ρ ob = (x -x ob ) 2 + (y -y ob ) 2 , ρ represents a safe distance between the robot and the obstacle. ρ ob represents the distance between the mobile robot and the obstacles, μ represents the gain coefficient of repulsive field. Outside this distance, the repulsion of the obstacle has no effect on the robot. When it is less than this threshold, the repulsion of the obstacle to the robot is affected by the distance between the two.
Introduction to TAPF For the APF, the core problem is local oscillation. This paper uses the Tangent Artificial Potential Field (TAPF) to solve the three-point-one-line problem in ordinary situations. When The obstacles mentioned in this article are all approximated as a circle, and the coordinates of the obstacles are (x ob , y ob ) and the radius of the obstacles is R. The following is the specific calculation formula: x o = x min + R y o = y min + R R = [(x max -x min )/2] 2 + (y max -y min )/2] 2 (6) where x max ,x min and y max ,y min are the maximum and minimum values of the horizontal and vertical coordinates of the obstacle in the coordinates respectively. The value range of the direction angle of the mobile robot is[-π, π], which is convenient to calculate the direction of the robot's movement. The formula is expressed as: φ(x, y) = arctan( y x ) x > 0 π + arctan( y x ) y >= 0, x < 0 π + arctan( y x ) y < 0, x < 0 π 2 y > 0, x = 0 -π 2 y < 0, x = 0 (7) It is also mentioned in this paper that when the robot, obstacle and target point are in a straight line, local oscillation will occur. To overcome this problem, we modify the repulsive force U 1 rep (x) by adding a new U 2 rep (x). Make a tangent to the obstacle from the position of robot, and the new input is along the tangent direction. The APF repulsion field formula U 1 rep (x, y) can be expressed as: U 1 rep (x, y) = μ 1 (x 0 -x) 2 + (y 0 -y) 2 -R (8) The angle φ 1 rep between the robot center point and the obstacle center point can be expressed as: φ 1 rep = φ(y o -y, x o -x) (9) According to the knowledge of geometry, there are two tangents from a point outside the circle (x, y) to a circle with a center point(x o , y o ) and a radius of R. Although there are two tangents, we only need to select one of them. The formula for calculating the slope of the tangent is as follows: k = x o y o + xy -xy o -x o y + R x 2 + y 2 + x 2 o + y 2 o -2x o x -2y o y -R 2 -R 2 + x 2 o -2x o x + x 2 (10) The horizontal and vertical coordinates of the tangent point x 1 = -ky+xo+k 2 x+kyo 1+k 2 , y 1 = kxo+y+k 2 yo-kx 1+k 2 , and U 2 rep (x, y) are expressed as : U 2 rep (x, y) = μ 1 (x 1 -x) 2 + (y 1 -y) 2 (11) Then we can calculate the angle between the robot and the tangent to the obstacle: φ 2 rep = φ(y 1 -y, x 1 -x) (12) Finally, the final form of U rep (x, y) can be calculated as follows. U x rep (x, y) U y rep (x, y) = U 1 rep (x, y) cos φ 1 rep U 1 rep (x, y) sin φ 1 rep + U 2 rep (x, y) cos φ 2 rep U 2 rep (x, y) sin φ 2 rep (13) The above situations are all aimed at the three-point collinear problem, but the actual situation is far more complex than the three-point collinear problem. Especially for semiclosed obstacles, The TAPF cannot solve this problems.
Introduction to the principle of jumping out of semi-closed obstacles by BVO-APF algorithm The walking-along-the-wall strategy and the technique of virtual target points are two methods that have been suggested for semi-closed obstacles. Although this issue can also be resolved, the robot will take more steps as a result. So, a BVO-APF algorithm is suggested in this study. The algorithm's main idea is that the robot can return to its original path and escape the semi-closed obstacle, as shown in Figure 4 . The semi-closed obstacles are sealed using the virtual obstacle technique. Now the robot is facing a totally enclosed obstacle, we can think of it as an obstacle with a large radius. The robot can then use the tangent algorithm to effectively hop out of the semi-closed obstacles. There is an included angle θ between the coordinates of the two center points. When the robot does not enter the semi-closed obstacle, the angle θ is θ + . When the robot is parallel to the center point of the most marginal obstacle, the angle θ is 0 , and when the robot enters the obstacle, the angle is θ -. When the mobile robot enters the semi-closed obstacle, local oscillation will occur. The mobile robot is stationary or oscillated somewhere in the semi-closed obstacle, and the mobile robot will calculate the angle θ . If it is inside the semi-closed obstacle, the mobile robot will retreat according to the moving path when it came in. The included angle θ between the two will gradually decreases. When the included angle θ from θ -to θ + , it will show that the mobile robot withdraws from the obstacle. Fig. 5 The angle diagram of the mobile robot before and after entering the obstacle In order to make the robot escape from the semi -closed obstacles smoothly, we define the included angle θ between the two center points, and the formula is as follows: θ = θ -= -cot( ρ 2 obi +ρ 2 ob(i-1) -ρ 2 i 2ρ obi ρ ob(i-1) ) inside θ + = cot( ρ 2 obi +ρ 2 ob(i-1) -ρ 2 i 2ρ obi ρ ob(i-1) ) outside (14) where ρ obi represents the distance from the current position of the mobile robot to the obstacle at the edge of the semi-closed obstacle, ρ ob(i-1) represents the distance between the current position of the mobile robot and the previous position of the mobile robot.The expressions are as follows: ρ obi = (x -x ob ) 2 + (y -y ob ) 2 (15) ρ ob(i-1) = (x i-1 -x ob ) 2 + (y i-1 -y ob ) 2 (16) where (x ob , y ob ) represents the coordinates of the center point of the edge obstacle, (x i-1 , y i-1 ) is the position of the previous step of the mobile robot. At this time, the robot judges the included angle θ between the mobile robot and the edge obstacle. If θ is less than 0, it is deemed that the obstacle has not been withdrawn; if the included angle θ is greater than 0, it is deemed that the obstacle has been withdrawn. When the mobile robot completely withdraws from the semi-closed obstacle, it will search the position of two edge obstacles, and the system will seal the semi-closed obstacle by adding virtual obstacles. Then jump out of the whole obstacle by using the tangent algorithm. Generally, if the radius of the obstacle is large, the included angle θ is greater than 0, but the mobile robot does not completely jump out of the semi-closed obstacle. Fig. 6 Obstacles with large radius As shown in Fig 6 , in some obstacles with large radius, although the above conditions are met, the mobile robot still does not fully jump out, so we change the judgment condition of θ to θ 1 , the formula of θ 1 is as follows: θ 1 = θ + + N Rδ ( 17 ) where δ is the angle of adjustment, R is the radius of the edge obstacle and N is the adaptive adjustment parameter. When the radius of the edge obstacle is larger, the judgment threshold of θ will increase, which will ensure that the mobile robot can completely withdraw from the obstacle. When θ is greater than 0 and θ is greater than θ 1 , it is defined as the mobile robot jumping out of a semi closed obstacle. The environment awareness function package is mainly responsible for driving the sensor and obtaining real-time environmental information around. The sensor source is generally radar. However, with the development of depth camera, the depth camera can also obtain environmental information and calculate the depth of field to achieve the effect of radar. The odometer is mainly realized by the robot's positioning function package, which is actually composed of a series of sensors, such as using an encoder to collect the robot's linear velocity, gyroscope and accelerometer to calculate the robot's attitude, and compass to locate the robot's direction. Adaptive Monte Carlo localization is an algorithm developed during the development of robot, which can make the localization information of robot more accurate.
BOV-APF algorithm flow chart However, this algorithm is optional, because the basic data fusion algorithm can also realize the positioning function. When the robot moves in an unknown environment, the map server can not be used. The map server will use the odometer and sensor together only when moving in a known environment. However, when these function packages implement each function, the coordinate system of each function package is different. In order to be used together, sensor coordinate conversion is required.
ROS Map establishment This paper uses the laboratory as the background to make local and global path planning maps respectively. The physical map and physical Trolley are shown in Figure 18 and Figure 19 . Using the gmapping algorithm, and then using the keyboard to control the robot to move in the real environment, and finally build a global map. The effect of the map is shown in As future work ,the first overarching work is to apply the method in the paper to an environment with dynamic obstacles. The second task is to avoid the trolley from entering the local oscillation point as much as possible. Finally, the global path should be as short and smooth as possible while ensuring that the mobile robot can reach the target point. Fig. 1 1 Fig. 1 Schematic diagram of APF
Fig. 2 2 Fig. 2 Local Oscillation of the Three-point-one-Line Problem
y) is the real-time coordinate position of the robot, (x o , y o ) represents the coordinate position of the target point, ρ 1 represents a limit distance between the robot and the target point. n, k(k > 1) and m are adaptive parameters, the value of k depends on the distance between the starting point and the target point.
Fig. 3 3 Fig. 3 Local Oscillation of the Three-point-one-Line Problem
Fig. 4 4 Fig. 4 Semi-closed obstacle path map
Fig. 7 7 Fig. 7 BOV-APF algorithm flow chart
Fig. 8 Fig. 9 Fig. 10 8910 Fig. 8 Experimental diagram of traditional APF
Fig. 11 11 Fig. 11 Path comparison of three algorithms in triangle obstacle
Fig. 12 12 Fig. 12 Path comparison of three algorithms in U-shaped obstacles
Fig. 14 APF 14 Fig. 14 APF Gravitational and repulsive force changes
Fig. 15 TAPF 15 Fig. 15 TAPF Gravitational and repulsive force changes
Fig. 16 16 Fig. 16 BVO-APF Gravitational and repulsive force changes
Fig. 17 17 Fig. 17ROS Navigation Framework
Fig. 18 18 Fig. 18 Local topographic map
Figure 20 . 20 Figure 20. We control the robot to generate the renderings of Figure20and Figure21, and then we put the BVO-APF algorithm proposed in this paper on the robot for verification.
Fig. 20 20 Fig. 20 Generated local path map Fig. 21 Generated Laboratory Corridor Map
Fig. 22 Fig. 24 Fig. 25 222425 Fig. 22 Triangular obstacle path diagram for BVO-APF
This research was partially funded by the National Natural Sciencce Foundation of China under grant 62073223, the National Natural Sciencce Foundation of China under grant 11502145, the China national R&D Key Research Program under grant 2020YFC227502 and the National Natural Sciencce Foundation of Shanghai under grant 22ZR1443400.
Table 1 1 the table of problem 1 Algorithm APF TAPF BVO-APF Starting point (0,0) (0,0) (0,0) Target (44,55) (44,55) (44,55) Stop point (44.3,55.4) (44.3,55.3) (44,55) Obstacle coordinates (42,52) (42,52) (42,52) Situation Failure Failure Success
Preprint submitted to Journal of Field Robotics December 24, 2022
|
10.1007/s10803-020-04816-6
|
cc-by
| null | null |
openalex
|
The impact of the 2019 coronavirus pandemic in the United States is unprecedented, with unknown implications for the autism community. We surveyed 3502 parents/caregivers of individuals with an autism spectrum disorder (ASD) enrolled in Simons Powering Autism Research for Knowledge (SPARK) and found that most individuals with ASD experienced significant, ongoing disruptions to therapies. While some services were adapted to telehealth format, most participants were not receiving such services at follow-up, and those who were reported minimal benefit. Children under age five had the most severely disrupted services and lowest reported benefit of telehealth adaptation. Caregivers also reported worsening ASD symptoms and moderate family distress. Strategies to support the ASD community should be immediately developed and implemented.The closure of schools, clinics, and community programs puts children with autism spectrum disorder (ASD) at increased risk for negative outcomes. In addition to special education instruction, schools commonly provide critical services to children with ASD, including speech and language therapy (SLT), physical and occupational therapy (PT/ OT), behavioral interventions and psychological supports (Koegel et al. 2012) . Additional services targeting younger children, including interactive play-based therapies and Applied Behavioral Analysis (ABA), typically involve many hours (e.g. 20-40 h per week) of face-to-face intervention in the clinic or school and at home (Behavior Analyst Certification Board 2014) . Children with ASD are also more likely than the general population to utilize clinic-based medical and mental health services (Cidav et al. 2012; Brookman-Frazee et al. 2011) . Previous studies, before COVID-19, have established that the majority of school-age children with ASD (83-96%) in the United States were receiving some type of service or therapy (Monz et al. 2019; Zuckerman et al, 2017) . The delivery of ASD services and therapies in an online or remote format may partially ameliorate the impact of COVID-19. Prior research indicates that telehealth services may be an effective means of providing medical and psychiatric care to children with ASD (Bearss et al. 2017; Parsons 1 3 et al. 2017) in addition to increased convenience for and empowerment of caregivers (Wallisch et al. 2019) . Online parent-training programs may also be effective for therapies delivered in the home by a caregiver. A small study of parent-mediated therapies suggests that there are no differences in outcomes when parents are trained in-person versus via remote platforms (Hao et al. 2020 ) and that parent training delivered remotely can be effective at reducing problem behaviors in children with ASD (Lindgren et al. 2020) . Overall, however, there is little evidence supporting the successful delivery of ASD services (including special education) in an online or remote setting. The disruption of critical services and therapies, along with other changes in daily life, may result in worsening autism symptoms, increased behavioral challenges and decreased mental well-being for children with ASD. Prior research has shown that parents/caregivers of children with ASD have higher levels of stress than parents/caregivers of typically developing children (Hayes and Watson 2012) and with other neurodevelopmental disorders (Valicenti-McDermott et al. 2015) . Furthermore, caregiver stress has been shown to increase with the severity of a child's ASD symptoms (Argumedes et al. 2018; Shepherd et al. 2018) . The COVID-19 pandemic may place parents/caregivers under additional stress and risk poorer mental health outcomes and family crisis. Here we describe the impact of the COVID-19 pandemic on families of children with autism enrolled in the Simons Powering Autism Research for Knowledge (SPARK) research study. SPARK is a national, online research cohort of individuals with ASD and their family. We surveyed caregivers of children (and dependent adults) with ASD enrolled in SPARK to report disruptions in services and therapies for ASD, frequency of transition to online/telehealth and perceived benefit, and the overall emotional wellbeing of parents and children with ASD during the early months of the pandemic in the US.
Methods
Participants Participants were recruited from SPARK, an online research cohort of individuals with ASD and their immediate family members (The SPARK Consortium 2018). The participants were caregivers (primarily parents) who initially registered their family into SPARK. All ASD diagnoses for dependent children in SPARK are parent/caregiver reported. Previous studies have demonstrated that parent/caregiver report diagnoses in an online research cohort are highly reliable (Daniels et al. 2012; Lee et al. 2010) . For multiplex families, a single child was selected at random, and the parent was asked to complete the survey with this child in mind.
Procedures The SPARK study is approved by the Western Institutional Review Board (WIRB). All participants who consent to join the SPARK study agree to be contacted to complete additional questionnaires. Participants received an email invitation and up to two reminder emails from March 20, 2020 through April 1, 2020, for the first COVID-19 questionnaire (final participants completed on April 3, 2020), and April 23, 2020 through April 29, 2020 for the follow-up questionnaire (final participants completed on April 30, 2020). Only participants who completed the first questionnaire (N = 9030) were invited to complete the follow-up questionnaire. No incentive was provided for participation, but SPARK developed an infographic and article to share results with participants (Simons Foundation 2020).
Measures With the exception of the Brief Family Distress Scale (BFDS), all items in the questionnaires used by this study were written by the authors specifically to assess the impact of COVID-19 on the autism community. Questionnaires are included in supplemental files, with questions used as part of this study highlighted in bold text. The BFDS was used to measure the subjective experience of crisis. Developed for use in families of children with ASD, this single-item questionnaire asks parents to indicate their level of distress on a scale from 1 'everything is fine we are not in crisis' to 10 'we are currently in crisis and it could not get any worse.' Scores indicate caregiver impairment to effectively cope with or respond to current stressors. Impairment is categorized as 'none' (scores 1-3), 'moderate' (scores 4-5) or 'marked' (scores 6-10). Families with marked impairment are considered to be 'near or in crisis.' In a reference population of caregivers, the BFDS was normally distributed with a mean of 4.28 (SD = 1.65) (Weiss and Lunsky 2010) .
Data Analysis Analyses included measures of central tendency (e.g. means and proportions) as well as tests for differences (e.g. chisquare and one-way analysis of variance tests) across the following age categories: 5 years and younger; 6 to 17 years; and 18 years and older. The Fisher's exact test, as compared to the Chi-square test, was used whenever a cell size in any of the tabulations across age categories was less than five. The survey sample was also compared with the SPARK population on all sociodemographic and clinical characteristics 1 3 to identify any significant differences. Data were analyzed using Stata version 12.1.
Results Among parents or caregivers who completed the first survey (N = 9030), 4369 completed the second survey. Among those, 760 reported receiving no services at baseline and were excluded. An additional 107 participants were excluded due to the inconsistency of their responses. In the followup survey, these participants reported receiving services at baseline. However, when they were asked whether services were impacted, their response was "Not applicable, my child doesn't receive ASD services or therapies." The final sample included 3,502 parents and caregivers of dependent children and adults with ASD who completed both baseline and follow-up surveys and reported receiving services at baseline, representing 39% of all families that completed the initial survey (Table 1 ). Compared to invited participants, survey respondents were slightly older (43 years vs. 41 years, p < 0.001), more likely female (93% vs. 90%, p < 0.001) and White (80% vs. 68%, p < 0.001), and less likely Hispanic (16% vs. 20%, p < 0.001). Respondents were also more likely to come from the Northeastern (19% vs. 16%) than the Southern region United States (31% vs 37%, p < 0.001) and to report household incomes over $100 K (31% vs. 26%, p < 0.001) compared to non-respondents. There was no significant difference in child sex at birth comparing survey respondents to invited participants, however children included in this study were slightly older (11.8 vs. 11.4 years, p < 0.001). While mean Social Communication Questionnaire (SCQ) scores did not differ between the two groups, children included in the study were more likely to report intellectual disability (21% vs. 19%, p < 0.001) and use of longer sentences (56% vs. 53%, p = 0.003). SPARK participants included in this study were comparable to those who completed only the first COVID survey, with few exceptions. Study participants were slightly older and children with ASD were slightly younger. Parents of ASD children were also more likely to report no speech or fewer words than those who participated in the first survey. Lastly, those included in this study had slightly higher overall household income and were more likely to come from a large metropolitan area. While the aforementioned differences were statistically significant (at p < 0.05), the magnitude of these differences was not considerable.
Baseline Services and Disruptions Due to COVID-19 Irrespective of age category, the majority of parents and caregivers reported disruptions to special education (80%), 1 3 speech and language therapy (SLT; 88%), physical and occupational therapies (PTOT; 84%), and applied behavior analysis (ABA; 77%) (see Fig. 1 for summary by age category). There were no significant differences across age groups in the most commonly received and disrupted services at baseline-special education, and SLT. In contrast, PTOT and ABA services were significantly more disrupted among the preschool-age group, as compared to school-age children and dependent adults (PTOT: 87% vs. 83% and 71%, respectively; ABA: 79% vs. 77% and 62%, respectively). Fewer than half of all parents/caregivers reported disruptions to medical services (39%).
Use and Benefit of Remote and Online Services at Follow-Up In general, the majority of individuals with ASD across all age groups did not receive online/adapted services in most categories at follow-up (Fig. 2 ). Among all remote and online services offered, only two-mental health and medical services, were reported by a majority of parents and caregivers of all children and dependents as significantly or moderately beneficial (55% and 63%, respectively). A considerably greater proportion of dependent adults (68%) reported significantly or moderately benefitting from online mental health services as compared to school-age children (54%) and preschool-age children (22%) (Fig. 3 ). School age children were significantly more 1 3 likely to report a significant or moderate benefit to special education and SLT services offered remotely or online, as compared with preschool-age children and dependent adults (special education: 40% vs. 16% and 39%, respectively; SLT: 45% vs 36% and 37%, respectively). Lastly, fewer than half of all respondents reported a significant or moderate benefit to remote or online PTOT (36%) and ABA (44%), and there were no significant differences across age groups.
Impact on Families Regarding impact of COVID-19 on child behaviors and parent stress and distress, just under two-thirds (64%) of all parents and caregivers reported that disruptions to services and therapies had severely or moderately impacted their children's ASD symptoms, behaviors or challenges, and no differences were observed across age groups (see Table 2 ). While three-fourths of all parents reported extreme or moderate stress due to disruptions in their children's services and therapies, it was greatest among preschool age children, (80%), followed by school age (73%) and dependent adult children (68%; p < 0.001). Parents of school age children were significantly more likely to endorse greater distress, measured both quantitatively and categorically, as compared to preschool-age and dependent adult children [mean 3.7 (SD 1.5) vs. 3.6 (1.5) and 3.4 (1.4), respectively]; categorically, as marked impairment: 10% vs. 8% and 5%, respectively).
Discussion The aim of this study was to examine the impact of COVID-19 on ASD services and caregiver perception of effectiveness of online adaptation, and impact of service disruption on families. Using previous studies as an approximation of service levels before COVID-19 (Monz et al. 2019; Zuckerman et al. 2017 ), this study found extensive disruptions to all types of ASD services due to COVID-19 at the beginning of the pandemic. Disruptions were more commonly reported for high intensity (e.g. daily or weekly) services such as ABA, special education, SLT, and PT/OT. Medical services were the least disrupted. This may be due to less frequent use, greater variability in the closing of medical offices (e.g. many remained open but with adapted procedures to limit person-to-person contact), and/or greater pre-COVID-19 familiarity with and/or more rapid transition to telehealth services. Most services were disrupted for a majority of individuals with ASD across all age groups, however, suggesting a widespread negative impact of COVID-19 on services and therapies. Individuals with autism and their families are an important vulnerable group for consideration of adapted services and additional support during similar emergency situations. One solution to ameliorate social distancing impact on ASD services and therapies is to transition to telehealth or online platforms, which allow for continuity of care without increasing COVID-19 risk. At follow-up, all service types were adapted to telehealth delivery for some participants; however, for most services only a minority of dependent 1 3 children/adults were receiving them in this manner. The exception was for mental health services, although this type of service is likely more easily adapted to telehealth delivery. Overall, the reported benefit of online and telehealth services was low (< 50%) across all service and therapy types and age groups except for medical and mental health services and dependent adults who receive ABA. Many traditional evidence-based therapies and services involve interactive play, peer-to-peer interactions, and reinforcers which are harder to deliver remotely. Families of preschool-aged children reported the least overall satisfaction with online and telehealth therapies, with a majority of participants reporting little to no perceived benefit for services such as ABA, SLT, and PT/OT. Adapting services to a telehealth format for this age group may be particularly challenging given the need to maintain the child's attention on a computer screen. Many of these parents and children may also be relatively new to therapy, which would present distinct challenges when the therapist cannot be physically present with the family. Disruption to services and therapies is an additional stressor on families with a child or dependent with ASD during an already stressful time due to the COVID-19 pandemic. A majority of dependents with ASD at all ages experienced worsening ASD symptoms. Increased ASD symptoms, some of which may be extreme (e.g. aggression, self-harm), along with skill loss or stagnation are likely to be stressful for both child and family. While a large majority of parents reported extreme to moderate stress specifically due to the loss of services and therapies, parents of preschool aged children were most stressed. This may be due to the emphasis on skill acquisition at this critical developmental period, with lost time being perceived as more impactful for this group. Parents/caregivers of school-aged children reported higher levels of overall distress than those of dependents of other ages. A higher percentage of older children have co-occurring mental health conditions, such as attention deficit/hyperactivity disorder (ADHD), anxiety, and disruptive behavior disorder (Neumeyer et al. 2019) , that would be exacerbated by increased stress, which may contribute to challenges for this age group. It is notable that nearly 50% of families surveyed are endorsing distress at a level of moderate/marked impairment. This increased distress may put these families at higher risk of acute crisis and warrants monitoring by clinicians and other service providers in contact with these families as well as attempts to develop and share resources that may increase coping and reduce stress. Overall, families with a child or dependent with ASD are experiencing major service disruptions and reporting feeling increased stress/distress due to the COVID-19 pandemic. Even when services and therapies are adapted into an online or telehealth format, many parents/caregivers report that these adaptations are not successful for a majority of therapy types. Additional research is needed to develop interventions that can be adapted and delivered remotely to various age groups. Such efforts may have the added benefit of helping to address disparities documented in rural and other underserved communities (Monz et al. 2019) . Service providers should also consider solutions that allow for some in-person contact for critical therapies in a way that reduces risk, such as mask-wearing, contact tracing, and limiting in-person sessions to specific therapists/families, rather than rotating therapists or larger groups, to control the risk. Finally, clinicians and other service providers should monitor families with a dependent with ASD to watch for signs of worsening crisis risk and provide resources targeting areas of identified stress. Additional research and feedback from families may be needed to ensure that resources address the unique needs during a widespread emergency like the COVID-19 pandemic.
Limitations All responses are parent-reported and not assessed directly by a clinician or service provider. Because of the limited research in this area, the survey used in this study was not a validated measure, and therefore the results should be interpreted with caution. Studies used to approximate rates of service utilization among children with ASD before COVID did not involve the same participants, nor did they survey dependent adults with ASD, so they cannot be used as a true baseline comparison. Additionally, our sample is slightly overrepresented for higher income families and majority white, with under representation of African American, Asian, Native American, and Native Hawaiian compared to data from the U.S. Census Bureau (Humes et al. 2011) . While Hispanic respondents comprised 16% of the sample, which is representative of the US population, SPARK's requirement of English language fluency means that non-English-speaking Latinx or other families would also not be represented. Compared to participants who only completed the first survey, parents who completed both surveys were slightly more likely to come from a large metropolitan area, had a slightly higher household income, and had a less verbal child. Evaluation of stress, distress, and areas of need would benefit from additional research with broader outreach to identify the groups most impacted and how to ameliorate negative effects and effectively intervene during this challenging time. Table 1 1 Participant characteristics (N = 3502) Characteristic Parent/caregiver characteristics Age in years, mean (SD) 43.4 (8.8) Sex at birth, N (%) Male 234 (7) Female 3268 (93) ASD diagnosis, N (%) 40 (1) Child/dependent demographic and clinical characteristics Age in years, mean (SD) 11.8 (6.6) Sex at birth, N (%) Male 2797 (80) Female 705 (20) Hispanic, N (%), n = 3249 510 (16) Race, N (%), n = 3428 White/Caucasian 2599 (80) Black/African-American 145 (4) Asian 74 (2) Native-American 13 (0) Native-Hawaiian/Pacific Islander 2 (0) Other 98 (3) Multiple 317 (10) SCQ score, mean (SD), n = 2999 22.3 (7.0) Ever intellectual disability, N (%) 739 (21) Language level, N (%) No words/does not speak 465 (13) Uses single words meaningfully 471 (13) Combines three words together into short sentences 591 (17) (for example, to request) Uses longer sentences of his/her own and is able to 1975 (56) tell you something that happened Sociodemographic characteristics Household income, N (%), n = 3497 $35 K and less 630 (19) Between $36 and $65 K 682 (20) Between $65 and $100 K 816 (23) More than $100 K 1,101 (31) Prefer not to answer 268 (8) US Census region, N (%), n = 3379 Northeast 662 (20) Midwest 1044 (31) South 814 (24) West 859 (25) Metropolitan classification, N (%), n = 3350 Large central metropolitan area 927 (28) Large fringe metropolitan area 993 (30) Medium metropolitan area 691 (21) Small metropolitan area 231 (10) Micropolitan area 260 (8) Non-core 158 (5) Urban/rural status, N (%), n = 3350 Urban 2932 (88)
Table 1 ( 1 continued) Characteristic Rural 418 (12) Fig. 1 Children/Dependents with disrupted ASD services/therapies due to COVID-19. Percentages are among those receiving the service at baseline. The number of children/dependents with disrupted services for each age category is provided. Significant differences across age categories, as per Chi-square or Fisher's exact test, are noted *p < .05 Fig. 2 Children/dependents receiving online or remote ASD services/ therapies at follow-up. Percentages are among those receiving the service at baseline. The number of children/dependents receiving online or remote services for each age category is provided. Significant differences across age categories, as per Chi-square or Fisher's exact test, are noted. *p < .05, p < .001 Fig. 3 Children/dependents benefiting from online or remote services/therapies. Percentages are among those receiving online or remote services at follow-up. The number of children/dependents reporting moderate/significant benefits for each age category is provided. Significant differences across age categories, as per Chi-square or Fisher's exact test, are noted. *p < .05, p < .001
Table 2 2 Impact on families(N = 3502) All respondents 5 years and younger Between 6-17 years 18 years and older P-value (n = 3502) (n = 603) (n = 2488) (n = 411) Child/dependent ASD symptoms (Q: To what extent have disruptions in services/ therapies negatively impacted your child's ASD symptoms, behaviors, or other challenges?) Severely/moderately 2151 (64) 382 (66) 1531 (64) 238 (62) Minimally/not at all 1203 (36) 201 (34) 856 (36) 146 (38) .531 Parent stress (Q: To what extent do you feel stressed or overwhelmed by the disruptions in your child's services/therapies?) Extremely/moderately 2475 (74) 470 (80) 1743 (73) 262 (68) Minimally/not at all 883 (26) 114 (20) 646 (27) 123 (32) < .001 Brief Family Distress Scale Mean (SD) 3.6 (1.5) 3.6 (1.5) 3.7 (1.5) 3.4 (1.4) < .001 No impairment 1747 (50) 314 (52) 1,199 (48) 234 (57) Moderate impairment 1442 (41) 239 (40) 1049 (42) 154 (38) Marked impairment 312 (9) 50 (8) 240 (10) 22 (5) .003
|
10.1371/journal.pbio.0040071
|
cc-by
| null | null |
openalex
|
Accurate auditory localization relies on neural computations based on spatial cues present in the sound waves at each ear. The values of these cues depend on the size, shape, and separation of the two ears and can therefore vary from one individual to another. As with other perceptual skills, the neural circuits involved in spatial hearing are shaped by experience during development and retain some capacity for plasticity in later life. However, the factors that enable and promote plasticity of auditory localization in the adult brain are unknown. Here we show that mature ferrets can rapidly relearn to localize sounds after having their spatial cues altered by reversibly occluding one ear, but only if they are trained to use these cues in a behaviorally relevant task, with greater and more rapid improvement occurring with more frequent training. We also found that auditory adaptation is possible in the absence of vision or error feedback. Finally, we show that this process involves a shift in sensitivity away from the abnormal auditory spatial cues to other cues that are less affected by the earplug. The mature auditory system is therefore capable of adapting to abnormal spatial information by reweighting different localization cues. These results suggest that training should facilitate acclimatization to hearing aids in the hearing impaired.Introduction Accurate auditory localization relies on the detection and interpretation of spatial cues generated by the interaction between sound waves and the head and external ears . Localization in the horizontal plane is based primarily on binaural disparity cues, interaural time differences (ITDs), and interaural level differences (ILDs), whereas spectral cues produced by the filtering properties of each external ear are used for making elevation judgments, discriminating between front and back, and, under certain conditions, for localizing sounds using one ear alone . The precise cue values corresponding to each direction in space depend on the physical dimensions of the head and ears. Acoustical measurements in different species have demonstrated that these dimensions vary from one individual to another , implying that listeners learn by experience to localize with their own ears. Indeed, psychophysical studies have shown that humans localize headphone signals that simulate real sound sources more accurately when these are derived from measurements made from their own ears than from the ears of other individuals [8, 9] . The plasticity of the auditory localization system can be probed by examining the capacity for adaptation to abnormal spatial cue values, produced either experimentally or as a consequence of pathological hearing. For instance, young barn owls [10, 11] and ferrets can adapt to the altered binaural cue values produced by long-term blocking of one ear and learn to localize accurately despite the presence of an earplug. Moreover, this behavioral compensation is mirrored by adjustments to the midbrain auditory space maps found in these species [13, 14] . Adaptive plasticity appears to be restricted, however, in older animals . Attempts to investigate whether humans can adapt to abnormal binaural cues have produced conflicting results . On the other hand, it is clear that adult humans can accommodate altered spectral localization cues . For instance, vertical localization accuracy gradually recovers over the course of a few weeks after disrupting these cues by inserting molds into one or both external ears . It remains to be seen whether the mature auditory system possesses a similar capacity to learn a new correspondence between binaural cues and directions in space, what conditions are required in order to evoke this plasticity, or what the underlying mechanisms might be. In this study, we examined the roles of behavioral training, vision, and error feedback in adaptation by adult ferrets to altered binaural cues produced by reversible occlusion of one ear. We show that rapid recovery of accurate spatial responses to sounds presented in the horizontal plane is possible when appropriate training is provided, that adaptation is not dependent upon feedback provided by the visual system, and that the basis for this plasticity involves learning to ignore the altered binaural cues and to attend selectively to those cues that are less affected by the earplug, namely lowfrequency ITDs and the spectral cues provided by the unoccluded ear.
Results We investigated the effects of monaural occlusion on the ability of adult ferrets to localize broadband noise bursts in the horizontal plane by measuring the accuracy with which they approached the source of the sound. Figure 1 shows data obtained from one representative animal following presentation of either 1,000-ms (Figure 1A Prior to occlusion of the left ear, this animal localized the longer duration stimuli perfectly, scoring 100% correct at all stimulus directions (Figure 1A and 1D ), but performed less well, particularly at lateral and posterior angles, at 40 ms (Figure 1G and 1J ). Plugging the left ear led to an immediate deterioration in performance at both stimulus durations (Figure 1B , 1E, 1H, and 1K). The largest errors were made on the left, the side of the earplug, where very few correct responses were observed. The stimulus-response plots (Figure 1B and 1H) show that the animal frequently mislocalized stimuli presented on the left to the opposite side of space. However, more errors were made at all speaker locations with the ear occluded, indicating the importance of binaural cues in this task. In order to examine whether localization accuracy could be relearned using the altered cues, the earplug was left in place and the animal retested every 6 d. The data obtained at 24 d revealed a marked improvement in localization accuracy (Figure 1C , 1F, 1I, and 1L). Performance for most stimulus locations on the right side, contralateral to the plugged ear, now approached that observed prior to the insertion of the earplug. Although relatively few correct responses were made to stimuli presented on the left (Figure 1F and 1L), these were now mostly lateralized to the correct side of space (Figure 1C and 1I), indicating that auditory localization accuracy had improved substantially since the left ear was first occluded.
Role of Vision in Auditory Adaptation It is well established that vision is used to coordinate spatial information provided by the different senses during development [14, . Moreover, it has been proposed that visual feedback about the accuracy of auditory localization could contribute to the adaptive changes observed after altering spectral cues in adult humans . We therefore compared the effects of monaural occlusion in three animals that had been raised normally and three others that had been visually deprived by binocular eyelid suture from infancy. This allowed us to determine whether visual feedback is required for adaptation of auditory localization in adult ferrets. We calculated the unsigned errors at each speaker location and, because of the distribution of responses shown in Figure 1 , combined these for the left and right sides of space. Data from individual animals are shown in Figure 2 . All six animals achieved a comparable level of performance prior to earplugging (session 0 in each panel of Figure 2 ), indicating that the loss of patterned visual cues did not impair their ability to localize sound. As expected from Figure 1 , the magnitude of the errors increased, particularly on the ipsilateral side, following insertion of the earplug, but then declined steadily as the animals were retested every 6 d. The performance of these animals was significantly worse at 40 ms than at 1,000 ms (F 1,4 1⁄4 32.43, p , 0.01) and when the left ear was occluded (F 1,4 1⁄4 199.72, p , 0.01). To test the effect of visual experience, we computed linear regressions of mean unsigned error, averaged across subject and speaker location on each side of space (y), versus testing session with the earplug in place (x) for each group and each stimulus duration (see Figure 2 for regression lines). On the side of the earplug (Figure 2A and 2B ), all four regressions yielded significantly negative slopes (normal, 40 ms: y 1⁄4 96.52-5.89x, R 2 1⁄4 0.50, p , 0.05; normal, 1,000 ms: y 1⁄4 74.12-6.09x, R 2 1⁄4 0.73, p , 0.01; visually deprived, 40 ms: y 1⁄4 82.01-3.98x, R 2 1⁄4 0.72, p , 0.01; visually deprived, 1,000 ms: y 1⁄4 58.80-3.24x, R 2 1⁄4 0.73, p , 0.01). Using the method for comparisons of regression lines described by Snedecor and Cochran , we found no difference between the rate at which sighted and visually deprived animals adapted to the altered binaural cues (1,000 ms: F 2,12 1⁄4 0.27, p 1⁄4 0.77; 40 ms: F 2,12 1⁄4 0.24, p 1⁄4 0.79). Our results show that adult ferrets can learn to reinterpret the abnormal localization cues produced by plugging one ear. Moreover, this process does not require a fully functioning visual system. When these ferrets were learning the task, they were rewarded for making a correct response. However, when the earplug was in place, the animals were rewarded for approaching any of the 12 sound sources. The observed improvement in localization performance shown in Figures 1 and 2 therefore indicates that error feedback is not necessary for adaptation. Indeed, no difference in the rate of adaptation was found when these animals were subsequently replugged and rewarded for making correct responses only (see following text).
Learning Is Driven by Auditory Training It is not clear from the data shown in Figure 2 or from previous studies of auditory spatial plasticity in humans whether the degree and rate of adaptation are determined by the duration of exposure to altered spatial cues or by the extent to which those cues are actually used to localize sound. We addressed this issue by examining the effect of soundlocalization training on the reacquisition of accurate spatial responses in a new group of six sighted ferrets. After the left ear was occluded, these animals were tested every day and their behavior reinforced by rewarding correct responses. Data from individual animals that were trained with either 1,000-ms noise bursts (n 1⁄4 3, Figure 3A and 3C) or 40-ms noise bursts (n 1⁄4 3, Figure 3B and 3D ) showed that the localization errors initially caused by the earplug steadily declined at all stimulus locations. These improvements were observed both within and between sessions. Comparison of the overall error magnitude at the beginning and end of the 10-d period of monaural occlusion revealed a significant improvement in performance at both 1,000 ms (Wilcoxon matched-pairs signed-rank test: p , 0.02 in each case) and at 40 ms (p , 0.05 in each case). In the data presented so far, auditory performance was assessed by training the animals to approach the source of the sound to receive a reward. This is the most common method used for measuring sound localization in animals [26, 27] . Because of the limited number of response alternatives available (in this case, 12 loudspeaker/reward-spout combinations), this is essentially a ''categorization'' task in which the subject presumably selects the stimulus location that most closely matches the actual perceived direction of the sound source . We eliminated the possibility that the ferrets were learning to associate particular stimuli with specific reward spouts for the following reasons. First, the broadband noise stimuli were generated afresh on every trial and, within a given session, presented at a range of sound levels that were varied randomly. Second, as reported above, the animals could adapt to the altered spatial cues regardless of whether they were rewarded for licking any of the 12 reward spouts or just the spout associated with the speaker from which the stimulus had been presented. Moreover, in addition to the approach-to-target responses, we obtained an absolute indicator of sound localization by measuring the accuracy of the initial unconditioned head-orienting movements made by the ferrets following stimulus presentation. Head-orienting errors also increased in magnitude in all ferrets after the left ear was occluded and then declined during the period of monaural occlusion (F 1,4 1⁄4 206.72, p , 0.01). We observed no effect of stimulus duration (F 1,4 1⁄4 2.897, p 1⁄4 1.64), with all animals showing some improvement during the period in which the ear was occluded. This is illustrated in Figure 3E and 3F for two of the animals that were tested daily (the same animals whose approach-to-target responses are shown in Figure 3A and 3B, respectively). Like the approachto-target responses, head-orienting accuracy with 1,000-ms noise bursts was disrupted primarily on the side of the plugged ear and recovered with further testing (Figure 3E ). The magnitude of the errors made in response to 40-ms stimuli increased on both sides when the ear was first occluded, but again declined with further testing despite the continued presence of the earplug (Figure 3F ). Before the ear was plugged, the latency of the head movements in response to 1,000-ms stimuli was 175 6 37 ms (mean 6 SD). The final head bearing before the animal left the start platform to approach the sound source was reached at 627 6 114 ms after stimulus onset. Consequently, the head was moving during these long stimuli. However, monaural occlusion had no effect on either head-orienting latency (F 2,14 1⁄4 2.67; p 1⁄4 0.10) or the time taken for this first head turn to be completed (F 2,14 1⁄4 0.84; p 1⁄4 0.92). The very close similarity between the accuracy of the approach-totarget and head-orienting responses with 1,000-ms noise bursts suggests that the animal's localization judgment was made during the early part of the stimulus and that once the head turn had been completed, this decision was not usually altered during the remainder of the stimulus. Moreover, because adaptation was also observed with 40-ms stimuli, which were over well before the animal started to move, we can be confident that the steady improvement in auditory localization in ferrets experiencing abnormal spatial cues is not simply based on the animals learning to track the longduration sounds. Daily testing consistently resulted in faster and more complete adaptation than in the animals that were tested every 6 d (F 1,12 1⁄4 14.48, p , 0.01; Figure 4A ), even though the overall number of trials performed was the same in each group. By contrast, if after occluding one ear we waited for 6 wk before testing the animals again, we observed no improvement in their auditory localization scores, which remained at the same low level achieved when the earplug was first inserted (Figure 4A and 4B ). The consistency in the performance of these animals is also observed in normal ferrets tested over intervals of several weeks and indicates that they were able to remember how to do the task. However, adaptation to the altered spatial cues, and therefore an improvement in performance, within this time frame required the earplugged animals to be engaged in the sound-localization task. Furthermore, the extent and rate of improvement were determined by the frequency of training in using the altered localization cues rather than by the number of trials performed or the duration of monaural occlusion. In order to investigate the specificity of the training required to produce adaptation, we trained another group of ferrets to localize both auditory and visual stimuli, which were presented on different trials within the same session (see Materials and Methods). After collecting baseline data for each stimulus modality, we plugged the left ear and confirmed that their auditory localization ability was severely disrupted. We then provided the animals with daily training on the visual task alone for 10 d. At the end of this period, we retested their auditory localization ability and found this to be unchanged from that seen immediately after the earplug was inserted (Figure 4B ). This lack of adaptation is in stark contrast to the pronounced improvement in performance of the animals that received an equivalent amount of auditory training during this period (Figure 4B ). Learning in the sound localization task therefore appears to rely exclusively on auditory training, reinforcing our finding that adaptation is not dependent upon a visual recalibration of auditory space.
Aftereffect following Restoration of Normal Binaural Inputs If auditory-localization adaptation is based on a reinterpretation of the relationship between binaural cues and directions in space, restoration of a normal binaural input should produce an aftereffect toward the side of the previously plugged ear. Before the earplug was introduced, no bias was seen in the responses of the ferrets that subsequently adapted with daily training to altered binaural cues (Figure 5A -5C; p 1⁄4 0.49, Wilcoxon matched-pairs signedrank test). However, after the earplug was removed, the animals exhibited a bias toward the side of the previously plugged ear (Figure 5D -5F; p , 0.01). The aftereffect was particularly apparent for the frontal 1208; the errors made in this region of space were almost exclusively toward the left side (p , 0.01), as indicated by the dots below the diagonal line in Figure 5D-5F . An aftereffect was also observed for the animals that were tested every 6 d during a 42-d period of monaural occlusion. No bias in responses was observed in these animals prior to plugging (p 1⁄4 0.36), but after the earplug was removed, their responses were initially shifted toward the side of the previously plugged ear (p 1⁄4 0.01). Although consistently observed following auditory adaptation, the aftereffect was transient, usually disappearing after the first session following earplug removal, and was much smaller than the bias in the opposite direction produced by the introduction of the earplug (see Figure 1 ). Moreover, the changes underlying auditory adaptation persisted to some degree, since replugging several weeks later had a much less disruptive effect on localization performance (paired t-test for comparison of errors, p , 0.05 at both stimulus durations), which recovered more completely than when the earplug was first introduced (Figure 6 ).
Basis for Auditory Localization Plasticity The relatively modest behavioral changes observed following the removal and subsequent reinsertion of the earplug (Figures 5 and 6 ) imply that a systematic retuning of neurons to the altered binaural cue values is unlikely to provide the basis for auditory localization plasticity in the adult brain. The alternative possibility is that adaptation involves learning to place greater weight on other auditory spatial cues that are less affected by monaural occlusion. The earplugs used in this study attenuated sound across a broad range of frequencies (see Materials and Methods) and therefore introduced abnormal ILDs. It is also likely that they delayed the transmission of sound through the ear, particularly at low frequencies , and changed the ITDs corresponding to different sound directions. By contrast, the spectral localization cues provided by the open ear will have corresponded to their normal sound-source directions and therefore provided information that conflicted with the altered binaural cues. To examine the contribution of the open ear to the rapid adaptation observed with daily training following monaural occlusion, we modified the spectral cues provided by this ear by applying a unilateral mold that filled its various cavities and depressions while leaving a clear passage for sound to reach the external auditory ear canal (meatus; Figure 7A ). Reshaping the external ear in this fashion altered its azimuth directional transfer function (Figure 7B ), but had no effect on the ITDs available (Figure 7C ). Application of a unilateral mold had a small but nonetheless significant effect on the accuracy of horizontal localization in control ferrets (Figure 7D ; F 2,11 1⁄4 10.066, p , 0.01), consistent with this being based primarily on binaural cues. A much greater change was observed, however, when a unilateral mold was applied in order to reshape the open ear in ferrets that had previously adapted to an earplug. In these animals, localization accuracy was altered significantly by occluding the left ear and then, with the earplug still in place, by application of a mold to the right external ear (F 4,23 1⁄4 21.502, p , 0.01; Figure 7E ). Post-hoc tests revealed significantly lower scores when the left earplug was first inserted (p , 0.05), but no difference between preplug scores and those obtained following adaptation to the earplug. Applying a mold to the right ear then led to a marked impairment in performance (p , 0.01), which recovered as soon as the mold was removed (Figure 7E ). Localization performance was degraded by the unilateral mold at all stimulus locations but particularly on the ipsilateral (right) side. In fact, the mean scores obtained when a mold was applied to the right ear of animals that had previously adapted to a left earplug were no different from those recorded when they first received the earplug and were about half the magnitude of those made by the control animals in the presence of a unilateral mold. Because the acoustic measurements involved implanting a probe tube in the wall of the external auditory meatus under general anesthesia, we did not measure how the spectral cues were altered by the mold in each animal. However, in each of five ferrets that had learned to localize accurately with the left ear occluded, applying a mold to the right ear resulted in a marked increase in both the number and magnitude of the We further investigated this possibility by comparing the ability of a group of control ferrets with that of a group of animals that had adapted to a unilateral earplug to localize 1/6-octave-wide noise bursts centered on either 1 kHz or 15 kHz. Broadband noise bursts were used when these animals were learning to carry out the localization task and also when training the experimental animals to adapt to the earplug. By switching to narrowband stimuli once they had adapted, we were able to eliminate most of the spectral information but preserve binaural cues over restricted frequency ranges. The center frequencies were chosen on the basis of the ferret's head-related transfer function to provide ITDs at a value of 1 kHz and ILDs at 15 kHz. According to audiometric measurements, ferrets are almost equally sensitive to 1-kHz and 15-kHz tones , and both narrowband stimuli should have been above threshold at all sound levels used, even in the presence of the earplug. However, to ensure that any differences in performance between the two groups were not due to certain combinations of stimulus level and position being inaudible to the earplugged animals, we analyzed only the responses to stimuli presented at the higher levels of 70-84 dB sound pressure level. Overall, both groups of ferrets localized the narrowband noise bursts less accurately than the broadband noise (Figure 8 ; F 5,23 1⁄4 19.926, p , 0.01). However, a much greater reduction in performance was observed in the animals that had learned to localize broadband signals accurately in the presence of a unilateral earplug, with their narrowband scores being significantly lower than those of the control group (F 3,15 1⁄4 14.123, p , 0.01). These data therefore indicate that by adapting to a unilateral earplug, ferrets become more dependent on the localization cues specific to broadband sounds. Comparison of the performance at the two narrowband stimuli revealed that both control and earplugged ferrets localized sounds with a low center frequency more accurately than those with a high center frequency (Figure 8 ). However, this difference was particularly pronounced in the ferrets that had adapted to a unilateral earplug, whose highfrequency scores were only slightly above the level expected by chance. These data further suggest that in learning to localize broadband sounds in the presence of the earplug, these animals were no longer using high-frequency ILDs.
Discussion The acoustical differences arising from variations in the size, shape, and separation of the two ears indicate that the neural mechanisms underlying auditory localization have to be calibrated by experience of the spatial cues available to individual listeners. This plasticity is most pronounced during development, when the cues are changing in value as a natural consequence of growth of the head and external ears . Our findings confirm the results of behavioral studies in humans [16, 17, in showing that the mature auditory system is also capable of adapting to markedly altered spatial cue values, and demonstrate that this plasticity can be greatly accelerated if stimulus-specific behavioral training is provided. Indeed, the extent and rate of adaptation are determined by the frequency of training, indicating that training has more benefit if concentrated into a shorter period of time. Moreover, we found that auditory localization plasticity does not depend on vision or error feedback and involves learning to place greater weight on acoustic cues that are largely unaffected by the earplug. Our measurements of auditory localization were based on the initial head-orienting responses made by the ferrets following stimulus presentation, as well as the accuracy with which they then approached the sound source. Because we observed essentially the same pattern of adaptation with both measures of performance and with stimuli of different duration, we can rule out the possibility that the improve-Figure 6 . Effects of Replugging the Same Ear Mean (6 SD) auditory localization scores before the left ear was occluded (''Pre-plug'') and at the start (''Initial plug'') and end (''Final plug'') of the plugging period for the six ferrets that were trained every 6 d while the earplug was in place. Superimposed on these histograms are the values obtained from individual animals, as indicated by the different symbols. (A) 1,000-ms noise bursts. (B) 40-ms noise bursts. The performance of the ferrets was the same before the ear was occluded in both runs, whereas the initial deficit caused by the earplug was lower and the recovery in performance with the earplug in place more complete in the second than in the first run. DOI: 10.1371/journal.pbio.0040071.g006 ment in auditory localization in the presence of a unilateral earplug relies on subsequent head movements or the animals learning to track the sound. Behavioral studies in primates and carnivores have shown that ablation or inactivation of the auditory cortex impairs the ability of these animals to approach the location of a sound source (e.g., [28, ). By contrast, orienting responses, although often abnormal, are still present [28, 35, 36] and are generally thought to be mediated more by subcortical mechanisms. Given this dissociation, our finding that both measures of sound localization ability exhibit a comparable degree of plasticity implies that spatial processing at different levels of the auditory system can be affected by training. Other studies have shown that adult perceptual learning is associated with changes in the response properties of neurons in the auditory cortex . Experience-driven modification of subcortical circuits could potentially be mediated by descending cortifugal pathways, which Suga and Ma have proposed might be involved in learning-induced plasticity.
Basis for Adaptation Raising barn owls [13, 44] and ferrets [12, 14] with one ear occluded leads to a compensatory shift in the map of auditory space in the superior colliculus, with the result that its alignment with the visual map in this multisensory midbrain nucleus is preserved. In owls, this developmental plasticity involves a shift in the tuning of the neurons for binaural localization cues [45, 46] . Consequently, earplug removal shifted the auditory spatial receptive fields toward the side of the previously plugged ear [13, 44] . Although we observed a significant aftereffect following restoration of a normal binaural input, this was much smaller and more transient than expected had the animals formed a new association between the cues distorted by the earplug and sound-source location. Consequently, it is likely that adaptation involves learning to ignore the altered cues and instead attending more selectively to cues that are unaffected by the earplug. Direct evidence for this is provided by our finding that modifying the spectral cues provided by the open ear reduced (C) Variation in ITDs within the horizontal plane before and after the mold was fitted. (D) Mean (6 SD) correct-score percentages for four control ferrets before the mold was fitted (''Pre-mold''), after applying it to the right ear (''Right mold''), and following its removal (''Mold removed''). (E) Mean (6 SD) correct-score percentages for a group of ferrets (n 1⁄4 5) before the left ear was blocked with an earplug (''Pre-plug''), at the start of the 10-d plugging period (''Initial left plug''), and at the end of this period (''Final left plug''). Note that the scores improved with daily training to just below the level achieved prior to monaural occlusion. At this point (with the earplug still in place in the left ear), a mold was applied to reshape the right external ear (''Left plug, right mold'') and then removed the following day (''Left plug, right mold removed''). Superimposed on these histograms are the values obtained from individual animals, as indicated by the different symbols. DOI: 10.1371/journal.pbio.0040071.g007 the localization accuracy of ferrets that had adapted to occlusion of the other ear to the level observed when the earplug was first introduced. Because the same unilateral manipulation of the spectral cues had a much smaller effect on the performance of control ferrets, this result indicates that the ferrets had learned to use spectral cues for localization in a region of space, namely the horizontal plane, where they normally make relatively little contribution other than in resolving front-back ambiguities in the binaural cues [47, 48] . This is further supported by our finding that ferrets that had adapted to a unilateral earplug localized narrowband sounds, in which spectral cues are impoverished, particularly poorly compared to control animals. Behavioral evidence for plasticity in the neural processing of spectral localization cues is also provided by the observation that adult humans can relearn to localize in elevation within a few weeks of altering these cues by using molds to reshape either one or both external ears . The pattern of adaptation observed by Van Wanrooij and Van Opstal to a unilateral mold suggests that the auditory system can learn to use novel spectral cues independently for each ear in order to localize sound in the vertical plane. This is consistent with the possibility that ferrets adapt to a unilateral earplug by becoming more reliant upon the monaural spectral cues provided by the contralateral ear for localization in the horizontal plane. However, reshaping the external ear with a mold will also alter interaural spectral cues, and other evidence indicates that monaural cues are by themselves insufficient to account for the localization performance achieved by the earplugged ferrets. Horizontal localization using one ear alone is usually very poor in ferrets and humans , suggesting that some input from the occluded ear is required in order for adaptation to take place. Following adaptation, the animals were able to localize low-frequency narrowband noise bursts disproportionately more accurately than high-frequency sounds, indicating that the animals are likely to use ITDs for this purpose. It therefore appears that the rapid and pronounced behavioral adaptation revealed in this study does indeed involve a reweighting of different spatial cues. The animals learn to ignore misleading cues, specifically high-frequency ILDs, with their localization responses becoming dominated by a combination of spectral cues and low-frequency ITDs. The mechanism underlying this reweighting continues to be plastic once the earplug is removed, as shown by the initial disruption in localization performance when the same ear is occluded for a second time several weeks later. Nevertheless, reinserting the earplug resulted in significantly smaller errors, and more complete adaptation, than when it was first introduced, indicating that the physiological changes that take place during the initial phase of adaptation persist to some degree, so that subsequent disruption of binaural cues can be accommodated more effectively. Elucidation of the physiological basis for adaptation first requires the neural locus of plasticity to be identified. As discussed above, this form of behavioral plasticity is likely to involve both the auditory cortex and subcortical processing levels. Virtual acoustic space stimuli -which allow individual localization cues to be manipulated independently-could then be used to investigate whether neuronal changes in sensitivity to particular cues take place as an animal adapts to a unilateral earplug.
Feedback and Auditory Plasticity Auditory spatial learning is presumably driven by feedback about the difference between actual and perceived stimulus location. Visual cues provide a possible source of sensory feedback about the accuracy of acoustically guided behavior and could therefore guide the adaptive plasticity of the auditory localization system. Indeed, there is extensive evidence that vision can recalibrate auditory localization, both during infancy [14, 23, 24] and in later life [24, . Knudsen and colleagues have previously examined the role of vision in adaptation to altered binaural cues by young barn owls. Although monaural occlusion during early development can induce adaptive adjustments in the tuning of midbrain neurons to binaural cues in the absence of vision, these changes are less complete than those observed in sighted birds . Moreover, when deprived of vision, owls are unable to recover normal localization behavior following earplug removal . In addition to being able to localize sound just as well as control animals that could see, we found that the visually deprived ferrets adapted to an earplug just as readily. Thus, not only can accurate and even supranormal sound localization develop in the absence of vision, our results show that visual feedback is also not required for recalibrating auditory space in adulthood. It is possible that this reflects the plasticity of auditory spatial processing that results from a long-term loss of vision, and that the auditory adaptive capabilities of animals raised with an intact visual system might show more dependence on vision. Nevertheless, it is clear that auditory adaptation is possible in the absence of visual feedback. We also found that feedback about response accuracy was not required, either in sighted or visually deprived ferrets, in order to see a progressive recovery of auditory localization in earplugged ferrets. This is consistent with studies in humans that have shown that improvement through training in sensory discrimination tasks is possible without error feedback [62, 63] . It therefore seems likely that the remaining, unaltered auditory cues provide sufficiently reliable information to form a basis for the relearning of accurate sound localization by the earplugged ferrets and that stimulusspecific training enhances the neural processing of these cues.
Perceptual Learning and Auditory Localization We found that monaural occlusion for 6 wk without training did not result in auditory localization adaptation, despite daily exposure to normal animal-house sounds, as the animals localized just as poorly at the end of this period as they did when the ear was first occluded. In contrast, juvenile ferrets raised in exactly the same environment do adapt without training, as do adult animals if the altered auditory spatial cues are experienced for a much longer period . Thus, the conditions in which the animals were kept did provide sufficiently rich sensory stimulation to enable learning to take place. The principal finding of the present study is that, with stimulus-specific behavioral training, adaptive changes in auditory localization responses can occur in adult animals much more quickly, within a few days rather than months. A related finding has recently been made by Bergan et al. , who showed that greater plasticity is possible in the midbrain auditory space map of adult barn owls that are allowed to hunt live prey compared to birds provided with dead mice. This dependence on training implies that rapid adaptation to altered auditory spatial cues by adult ferrets represents a form of perceptual learning. Unlike other examples of perceptual learning, however, in which practice leads to improved discrimination by individuals with normal sensory inputs , our data show that training can be used to recover function following the introduction of abnormal inputs. Given that auditory training has been used to improve temporal processing and alleviate language impairments in children [71, 72] , these findings suggest that it should be possible to use sound-localization training to promote rehabilitation in patients whose perception of auditory space is compromised as a consequence of hearing disorders.
Materials and Methods Animals. Thirty-one adult ferrets were used in this study, and were housed either singly (males) or in pairs or larger groups (females) in standard laboratory cages, which contained nesting boxes, litter material, and various toys. The animals were allowed out of their cages several times a week, so that they could explore and play with each other and with other objects in the room in which they were kept. All procedures received local ethical committee approval and were carried out under license by the UK Home Office. Ear manipulations. The earplugs used in this study were always applied to the left ear. The animals were sedated with medetomidine hydrochloride (domitor, 0.15 mg/kg; Pfizer, Sandwich, United Kingdom) and the external auditory meatus occluded with a foam plug (E.A.R., Boulder, Colorado, United States). The concha of the external ear was then filled with Otoform-K 2 silicone impression material (Dreve Otoplastik, Unna, Germany) so that it was easy to tell that the earplug was in place. The sound attenuation produced by these earplugs has been estimated, using auditory brainstem response audiometry , to be ;40 dB across a range of 1-16 kHz. Previous behavioral measurements also showed a comparable level of attenuation at 500 Hz . Otoscopic examination was used to confirm that the ear canals were unobstructed both prior to and following monaural occlusion. In nine ferrets, we examined the effects of modifying the spectral cues provided by the external ear. To do this, the animals were sedated, a tube temporarily inserted into the ear canal, and Otoform-A/flex ear-impression silicone (Dreve Otoplastik) injected into the concha and other cavities of the right external ear. The tube was then removed, leaving a clear passage for sound to reach the ear canal. The acoustical consequences of the mold were determined in one ferret under general anesthesia 22 lg/kg domitor plus 5 lg/kg ketamine i.p.) by measuring the azimuth directional transfer function, as described in detail in Schnupp et al. and in Parsons et al. . Briefly, a polythene probe tube was inserted into the wall of the external auditory meatus and attached to a Sennheiser cartridge (KE 4-211-2) (Sennheiser, High Wycombe, United Kingdom). A metal bar was attached to the skull so that the animal could be supported from behind on a small table positioned at the center of an anechoic chamber. Acoustic signals comprised 32,768-point Golay codes, played and recorded at 100 kHz using Tucker-Davis Technologies (Alachua, Florida, United States) system-2 hardware, and delivered by a KEF T27 loudspeaker (KEF Audio, Maidstone, United Kingdom) mounted on a robotic hoop system. The signal recorded by the probe microphone was passed through an antialiasing filter (7-pole elliptic filter, F c 30 kHz) and digitized. The ITDs were extracted from the microphone signals by cross-correlation of the impulse responses after low-pass filtering at 4 kHz. The amplitude spectra of the transfer function were obtained for each loudspeaker position and the contributions of the speaker and microphone removed by subtracting the amplitude spectra (in dB) measured in the absence of the animal. The plots were then smoothed with a linear Gaussian filter and the directional transfer function calculated by subtracting the mean spectral transfer function (in dB) from each of the individual functions. This procedure, which removes the location-independent components of the spectral transfer function, including the microphone and speaker transfer functions, was carried out before and after a mold was applied to the external ear. Behavioral measurements. Details of the positive-reinforcement paradigm have been described previously [28, 30] . Briefly, waterrestricted ferrets were trained to initiate a trial by mounting a platform and licking a waterspout at the center of a circular testing arena. This triggered the presentation of a single burst of broadband noise (low-pass filtered at 30 kHz) from one of 12 loudspeakers equally spaced around the perimeter of the arena at 08 elevation. The output of each speaker was flattened digitally and matched for sound level. Sound levels were varied randomly within a session from 56-84 dB sound pressure level. Following stimulus presentation, the animal approached its source and received a water reward for licking the spout in front of the speaker from which the stimulus had been played. In the first earplugging experiment, the ferrets received an equal amount of water for licking any of the 12 reward spouts. Incorrect responses were followed by 3 correction trials in which the stimulus was presented at the same location, after which a continuous stimulus was presented (none of these trials was used to assess performance). All animals were trained to a similar degree and scored .90% correct with 1,000-ms noise bursts before any of the experiments started. In some experiments, visual stimuli comprising single light flashes were presented from a light-emitting diode mounted above each of the speakers in the frontal hemifield (6908). These animals were trained to localize both auditory and visual stimuli by randomly interleaving noise bursts from all 12 speaker positions with light flashes of matching duration from the seven light-emitting diodes in the frontal hemifield. The latency and accuracy of the initial head movements made by the animals were measured by tracking the position of a reflective strip attached to the head using a vertically mounted infraredsensitive camera and video contrast detection device (HVS Image, Harlow, United Kingdom). Our software registered the x-y coordinates of the reflective strip at the onset of the sound and sampled these values at a rate of 50 Hz for the next second. They were converted to an angle relative to the initial head position. The latency of the head movement was taken as the point when the head first moved in the same angular direction over three successive trials. The software assigned a final head bearing based on two criteria: the mean of the last three data points after the peak angular acceleration had been reached and the three points prior to a consistent change in head direction, indicating that the animal had moved off the central start platform. These two values were highly consistent. The accuracy of the orienting response was determined by the difference between the final head bearing and the direction of the sound source. All the raw data were fed into algorithms for subsequent analysis. Criteria (e.g., for head movement latency and final head bearing) were set prior to data collection, so that there was no opportunity for the person handling the data to influence the result. Similarly, the reward spout licked by the animal was registered by the software, which converted this to a correct-score percentage and error magnitude and direction for each trial within a given session. -1F) or 40-ms noise bursts (Figure 1G-1L) from each of 12 loudspeakers located at 308 intervals in the horizontal plane. These data are displayed both as stimulus-response plots, showing the distribution of responses made to each stimulus location (Figure 1A-1C and 1G-1I) and as polar diagrams in which the correct-score percentage is plotted as a function of speaker angle (Figure 1D-1F and 1J-1L).
Figure 1 . 1 Figure 1. Effects of Monaural Occlusion on Ferret Auditory Localization (A-C and G-I) Stimulus-response plots for a representative animal, showing the distribution of responses (ordinate) as a function of stimulus location (abscissa). The size of the dots indicates, for each speaker angle, the proportion of responses made to different locations. Correct responses are those that fall on the diagonal line, whereas all other responses represent errors of different magnitude. (D-F and J-L) Polar plots showing the percentage of correct responses made to each of the 12 speaker locations. Each polar plot corresponds to the stimulus-response plot immediately above it. (A-F) Auditory localization responses to 1,000-ms noise bursts. (G-L) Responses to 40-ms noise bursts. The animal was tested before (A, D, G, J), on the same day that the left ear was plugged (B, E, H, K), and then every 6 d afterwards. Only the data obtained after 24 d of continuous plugging are shown (C, F, I, L). DOI: 10.1371/journal.pbio.0040071.g001
Figure 2 . 2 Figure 2. Adaptation Does Not Require Visual Feedback Data from three normally sighted (open symbols) and three visually deprived (filled symbols) ferrets showing the magnitude of the unsigned errors averaged across speaker location in the left (A and B; side of plugged ear) and right (C and D; side of open ear) hemifields. Each symbol represents a different animal. Data are shown for the session prior to insertion of the earplug (session 0) and for eight sessions, carried out at 6-d intervals, with the left ear occluded (sessions 1-8). DOI: 10.1371/journal.pbio.0040071.g002
Figure 3 . 3 Figure 3. Effects of Daily Training on the Accuracy of Auditory Localization by Ferrets Fitted with Unilateral Earplugs (A) Magnitude of the unsigned errors made by one representative ferret (F0031) in response to 1,000-ms noise bursts. The errors have been averaged across speaker location in the left (plugged) and right (unplugged) hemifields. Data are shown for the session prior to insertion of the earplug, for eight daily sessions with the left ear occluded, and for the first session following plug removal. (B) Equivalent data for another ferret (F0075), whose performance was measured with 40-ms noise bursts. (C) Mean errors (across all speaker locations) at the start and end of the plugging period for three animals that were trained to localize 1,000-ms noise bursts. (D) Mean errors at the start and end of the plugging period for three different ferrets that were trained to localize 40-ms noise bursts. (E and F) Head-orienting errors (mean 6 SEM) for the same animals whose approach-to-target responses are shown in (A) and (B), respectively. DOI: 10.1371/journal.pbio.0040071.g003
Figure 4 . 4 Figure 4. Auditory Localization Adaptation Is Accelerated by Modality-Specific Training (A) Change in performance (averaged across all speaker locations) over time in three groups of ferrets with unilateral earplugs. Each symbol represents a different animal. No change was found in trained ferrets that received an earplug for 6 wk, but were tested only at the start and end of this period (n 1⁄4 3, blue symbols and regression line). Two groups of animals received an equivalent amount of auditory training while the left ear was occluded. Although the earplug was in place for less time, a much faster rate of improvement was observed in the animals that received daily training (n 1⁄4 3, black symbols and regression line) compared to those that were trained every 6 d (n 1⁄4 6, red symbols and regression line). (B) Mean (6 SD) auditory localization scores at the start (filled bars) and end (open bars) of the plugging period for the animals that received daily auditory training for 10 d (black symbols in [A]), another group of auditory-trained ferrets that performed a visual localization task every day while the left ear was occluded for the same period (n 1⁄4 3), and the three ferrets that received no training during a 6-wk period of monaural occlusion (blue symbols in [A]). The only significant improvement in sound localization was found for the earplugged animals that received daily auditory training (F 2,6 1⁄4 15.097, p , 0.01). DOI: 10.1371/journal.pbio.0040071.g004
Figure 5 . 5 Figure 5. Aftereffect following Earplug Removal (A-C) Stimulus-response plots for three animals prior to insertion of the earplug. The stimuli were 1,000-ms noise bursts. The earplugs were left in place for 10 d, during which the animals underwent auditory-localization training every day (responses shown in Figures 3 and 4). (D-F) Stimulus-response plots for the same three animals for the session immediately following plug removal. DOI: 10.1371/journal.pbio.0040071.g005
Figure 7 . 7 Figure 7. Effect of Manipulating Spectral Cues on the Accuracy of Auditory Localization (A) Drawing of a ferret's head to show the mold that was used to fill the cavities and depressions of the external ear. Note that a passage was left for sound to pass through the external auditory meatus to the eardrum. (B) Azimuth directional transfer function measured before and after the mold was fitted.(C) Variation in ITDs within the horizontal plane before and after the mold was fitted. (D) Mean (6 SD) correct-score percentages for four control ferrets before the mold was fitted (''Pre-mold''), after applying it to the right ear (''Right mold''), and following its removal (''Mold removed''). (E) Mean (6 SD) correct-score percentages for a group of ferrets (n 1⁄4 5) before the left ear was blocked with an earplug (''Pre-plug''), at the start of the 10-d plugging period (''Initial left plug''), and at the end of this period (''Final left plug''). Note that the scores improved with daily training to just below the level achieved prior to monaural occlusion. At this point (with the earplug still in place in the left ear), a mold was applied to reshape the right external ear (''Left plug, right mold'') and then removed the following day (''Left plug, right mold removed''). Superimposed on these histograms are the values obtained from individual animals, as indicated by the different symbols. DOI: 10.1371/journal.pbio.0040071.g007
Figure 8 . 8 Figure 8. Effects of Auditory Adaptation on the Ability of Ferrets to Localize Narrowband Noise Bursts Mean (6 SD) correct-score percentages for four control ferrets (gray bars) and four animals that had learned with daily training to localize broadband noise after blocking the left ear with an earplug (open bars). Data from individual animals are shown by the different symbols. The scores obtained following adaptation are shown for the broadband stimuli used for training and for 1/6-octave-wide noise bursts centered at either 1 kHz or 15 kHz. Note that while all animals achieved lower scores with the narrowband (NB) stimuli, the animals that had adapted to an earplug performed particularly poorly at the high center frequency. DOI: 10.1371/journal.pbio.0040071.g008
PLoS Biology | www.plosbiology.org
Plasticity of Spatial Hearing in Adults
|
10.21776/ub.jitek.2020.015.03.1
|
cc-by
| null | null |
openalex
|
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic compound of organic pollutants that is very persistent in the environment. This compound can accumulate in the body through contaminated food and can cause various adverse effects on human health. The purpose of this study was to observe the effect of casein tablets made from goat milk yogurt on Liver histopathological profile, residual TCDD and malondialdehyde (MDA) levels in broiler chicken exposed to 50 ng/mL TCDD. A total of 18 male broiler chickens aged 3 weeks were used in this study and were divided into 3 experimental groups based on a completely randomized design (CRD): 1 (negative control group), 2 (positive control group), 3 (therapeutic group) which were administered by 750 mg of casein from goat milk yogurt for 21 days. TCDD residue and MDA levels in the liver were measured using a UV-Vis spectrophotometer and thiobarbituric acid reactive substances test, respectively. The liver histopathological profile was observed using hematoxylin-eosin staining. The results showed that the administration of casein tablets decreased the level of TCDD residue and MDA by 5.75% and 43.74%, respectively. Moreover, the liver histopathological profile of the therapeutic group was better than that of the positive control group. It is suggested that casein tablet made from goat milk yogurt possesses anti-dioxin and antioxidant capacities.INTRODUCTION Animal food products are important contributors to humans as sources of animal protein, but 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and other types of dioxins exist in animal products and feed caused by environmental pollution from combustion, industrial waste, factory smoke, and vehicle pollution, and pesticides (Hayward and Bolger 2005; Kan and Meijer 2007) Dioxin compounds can enter the food chain and the body of living things. In humans, various extreme health effects have been linked to high exposure to dioxin compounds such as TCDD, which can result in disability and retardation of fetal and infant growth, reproductive disorders, chloracne, hormonal dysfunction, mood changes, reduced mental performance, diabetes, changes in white blood cells, tooth defects, endometriosis and the biggest effect caused by dioxin compounds is cancer. Thus, it is necessary to overcome free radicals that cause oxidative stress caused by exposure to dioxin compounds with food products that contain antioxidants (Pemberthy et al., 2016) . Casein goat milk yogurt is one of the food products having antioxidant capacity. Casein is a relatively short type of bioactive peptide that contains about 2-20 amino acids that can function as antioxidants, antiinflammatory, antihypertensive, antimicrobial, and anticancer properties which have a potential role in maintaining the health of the human body (Neha et al., 2012; Bamdad et al., 2017) . Antioxidants can prevent chronic disease such as cardiovascular disease and cancer due to oxidative stress, and abnormal inflammatory response. Peptide intake from natural foods can delay the onset of disease by reducing oxidative damage and proinflammatory responses (Nuttal et al., 1999; Geronikaki and Gavalas., 2006) Among other animal food sources, milk protein is considered to be very nutritious and has a food component with a balanced composition of essential amino acids, and has also been reported as a good source of bioactive components (Bamdad et al., 2017) . In goat milk there is a casein complex polymorphism, casein content in goat milk represents 74% of total milk protein, whey protein amount is almost 17% and the proportion of non-protein nitrogen compounds is 9%. Genetic polymorphism and casein frequency in goat milk populations have higher casein micelles than cow milk which explains better digestibility of goat milk and milk products than cow's milk (Verruck et al., 2019) . Of the total milk protein casein accounts for 80%, mainly from αS1 (~ 40% of total casein), αS2 (~ 10% of total casein), β (~ 35% of total casein), and κ (~ 5% of total casein) casein sub-unit. Thus, casein phosphopeptides derived from gastrointestinal and commercial proteases can act as multifunctional bioactive peptides (Bamdad et al., 2017) . The number of casein benefits of goat milk yogurt for body health and as a multifunctional bioactive peptide in reducing the number of free radicals, so in this research used a product casein yogurt goat milk to see residual levels of TCDD, MDA, and histopathological feature.
MATERIALS AND METHODS
Conventional
Casein Tablet Manufacturing The method used in making casein tablets is wet granulation. By preparing tools and materials, the composition of ingredients is needed in weigh according to calculations. Then the gelatin is dissolved in hot water. Ingredients such as casein, collidon 30, collidon Cl, and lactose are the deep phases mixed for 15 min. After mixing 5 mL of gelatin is dropped to form a solid mass. Then sifted using a mesh No. 18 sieve to get the same granule size. Granules produced from the sieve are put into the oven for 2 d. The external phase material was mixed magnesium stearate and aerosol. Then the inner and outer phase ingredients are mixed for 10 min and sifted using mesh No. 18. Weighed 500 mg per tablet in parchment. Pressing the tablet using a press machine. Then an evaluation of the results of casein tablet products was carried out by calculating the uniformity of weights and sizes, testing the friability and fictionality of tablets by using a friabilator at a speed of 25 rpm for 4 min to determine the fragility of tablets. Then disintegration to determine the disintegration time of the tablet by simulating a gastric solution (gastric fluid) that is as many as 6 tablets put in Eppendorf tube, then up and down in a medium of water at 37C. Crush time is calculated based on the most recently destroyed tablet. Crush time requirements for non-coated tablets are less than 30 min. The composition of Casein Tablets can be seen in Table 1 below
Preparation of Animal Model The preparation of experimental animals was carried out based on ethical approval that was approved by the UB research ethics committee with ethics approval no. 1189-KEP-UB. Male broiler chickens are divided into 3 treatment groups with a population of 6 animals each/cage. Before the treatment is carried out, the chickens are kept and adapted first to the condition of the cage and given ad-libitum feed and drink. Male broiler chickens aged 3 wk were treated in a group of experimental animals namely group I (negative control) which were only given standard feed and drink. Group II (positive control) chickens exposed to TCDD 50 ng/mL/kg of feed for 21 d. Group III (therapeutic group) chickens exposed to TCDD 50 ng/mL/kg of feed and treated with 750 mg of casein from goat milk yogurt casein tablet products or 3 tablets of casein goat milk yogurt for 21 d by oral gavage. Then surgery is taken to take chicken liver, part of the liver is put in 10% formalin solution to make histopathological features and some others are wrapped in plastic clips, then stored in the refrigerator (-20C) for 7 d as a sample for determining TCDD residue levels and samples for determining levels MDA
Measurement of TCDD Residual Levels in the Liver A standard curve of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was made. Furthermore, chicken liver samples were cut into small pieces, mashed using a mortar, weighed as much as 5 g, added 10 mL of technical toluene solvent, covered with plastic wrap and aluminum foil, then stirred and homogenized using a magnetic stirrer for 2 h at speed of 8000 rpm. Then allowed to stand for 3 h, filtered using Whatman 41 filter paper, then the residual results are extracted again in the same way. The results of the sample filtrate from 2 times the extraction were taken to measure the TCDD residue levels using UV spectrophotometry at a wavelength of 305 nm. This method is based on references from (Binh et al., 2014) with modifications.
Determination of Malondialdehyde (MDA) Levels in Liver Malondialdehyde (MDA) calibration curves were made. Furthermore, chicken liver samples are cut into small pieces, mashed using a mortar, then weighed as much as 0.2 g, put into Eppendorf centrifuge added 1 mL Physiological NaCl. Centrifuged for 20 min at a speed of 8000 rpm, supernatant piped as much as 200 μL into the test tube. Then added 500 μL of distilled water, 200 μL of TCA 10%, 500 μL of HCl 1 N, 200 μL of Na-TBA 1%, homogenized, then heated in a water bath at 100C for 20 min. Lifted and cooled at room temperature, then centrifuged for 10 min at a speed of 3000 rpm, pipette and absorbance measured using a UV-Vis spectrophotometry at a maximum wavelength of 532 nm (Singh et al., 2002) .
Preparation and Reading of Histopathological Illustration Histopathological of the chicken liver using HE staining with fixation process by inserting it in 10% formalin solution, dehydration stage using ethanol with concentrated concentrations of 70% for 24 h, 80% ethanol for 2 h, 90%, 95% and absolute ethanol for 20 min. Then purification is done by soaking the organs in xylol I solution for 20 min and xylol II solution for 30 min. Then the infiltration and embedding steps were carried out using liquid paraffin in the incubator at a temperature of 58C -60C. Next, the cutting is done by clamping the mold in the microtome, and the tissue is cut with a thickness of 5 μm. The preparations were stored in an incubator at a temperature of 38C -40C for 24 h, then deparaffinated ith xylol I and II solutions, rehydrated with 95% ethanol, 90%, 80%, 70% and washed with running water, then stained HE. Then the histopathological preparations are read by scanning the liver using a light microscope at magnifications of 100×, 200×, and 400× displayed on the monitor screen. This method is based on from (Swarayana et al., 2012) with modifications.
Data Analysis Data analysis was performed quantitatively using the Analysis of Variance (ANOVA) statistical test using Microsoft Office Excel and SPPS 16.0 software for windows. And continued with the Tukey Honestly Significant Difference (HSD) test α = 0.05%. And data analysis of liver histopathology overview was done descriptively.
RESULTS AND DISCUSSION Effect of Casein Tablet Made from Goat Milk Yogurt on Dioxin Residue Level in Broiler Liver Exposed to 2,3,7,8-
Tetrachlorodibenzo-p-dioxin The results of making a standard curve obtained by the equation y = 1.69 + 0.0408 with a value of R2 = 0.9806. The ANOVA statistical analysis results followed by the HSD Tukey test with a confidence level of 95% residual levels of TCDD in broiler chicken liver can be seen in Table 2 below: The results showed that 750 mg casein therapy from casein products of goat milk yogurt given for 21 consecutive days decreased the level of TCDD residues in 3-wk-old broiler chicken liver exposed to TCDD 50 ng/mL/1 kg of feed for 21 d. This is done by TCDD exposure at 3 wk because at that age broilers are can adapt, are strong, and no longer care to control the temperature and brooder, related for 21 d by adjusting the harvest time of broiler chickens, namely 5-6 wk of age. An increase in TCDD residue that occurs in the negative control to the positive control of the liver results in a buildup of toxins that are difficult to excrete because not to dissolve in polar solvents. TCDD is the compound most toxic member of dioxin from the planar class, halogenated by aromatic hydrocarbons. TCDD is a widespread environmental contaminant produced by various chemical reactions and combustion processes. Dioxin is a toxic compound and very stable against the degradation process by the environment and by biological processes so that it is persistent in the environment. Dioxins bind strongly to the organic matter in the environment and the can be found in the upper layers of the soil. Due to the lipophilic and hydrophobic nature of dioxin, it is difficult for the substance to be metabolized in the body. As a result, dioxin easily accumulates and biomagnification occurs through the food chain or food. Exposure to TCDD in humans and vertebrate animals is one of the risk factors for cancer, immune system disorders, reproductive disorders accompanied by abnormalities of fetal development, endocrine system disorders, damage to the liver, heart, and nervous system disorders. Exposure to TCDD in humans occurs through three main pathways, namely work accidental exposure and exposure to a contaminated environment. The pathway for contaminants to enter the human body is through the gastrointestinal, dermal, and transpulmonary absorption. Exposure to humans most often is through the secondary pathway, namely through food. After experiencing absorption, TCDD in the circulation will bind to chylomicrons, lipoproteins, and other serum proteins, then quickly distributed to various organs. TCDD toxicity is not based on the number of exposures (rate of exposure) but depends on the body burden because of the long halflife of dioxin. The half-life of dioxin in mice is 12-31 d, mice 11-24 d, hamsters 11-15 d, marmots 30-94 d, cattle 40-50 d, monkeys 391 d, and in humans for 11 y. Dioxin toxicity is based on the total dioxin toxic equivalency (TEQ) approach that expresses the toxicity of pure TCDD material (Schecter et al., 2006; Van den Berg et al., 1994) . Dioxin pollution in food is still not getting much attention, especially in Indonesia. Very little information and data on pollution and the negative effects of dioxin. In a study conducted by Sani (2015) on dioxin residue levels using GC-MS as many as 50 samples consisting of 20 samples from Slaughterhouses-Yogyakarta, 15 samples from RPH-Central Java and 15 samples from RPH-East Nusa Tenggara. The results of the analysis of the average total TCDD residue in samples from RPH-Yogyakarta reached 13,624.38 pg/g with a range of 4,496.66-20.642.40 pg/g higher than the samples from RPH-East Nusa Tenggara which reached 1,623.98 pg/g in the range of 0.83-6.471.07 pg/g. Total TCDD residues in samples from RPH-Yogyakarta reached 54,497.52 pg/g and samples from RPH-East Nusa Tenggara reached 6,495.9 pg/g (Sani, 2015) . The results showed that the TEQ value in broiler chicken samples far exceeded the maximum allowable TCDD residue. The standard for the maximum limit of dioxin contamination in food ingredients in force in Indonesia is set by the Food and Drug Monitoring Agency (BPOM). The maximum value of dioxin and furan contamination in processed food can be seen in the range of 3-4 pg/g.
Effect of Casein Tablet Made from Goat Milk Yogurt on Malondialdehyde (MDA) Level in Broiler Liver Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin MDA is a product of lipid peroxidation by free radicals in the body. Detection of MDA levels generally uses serum or organs as samples. In this study, the liver is used as a determination of MDA levels because the liver has a detoxification function. Toxic compounds that enter the body will undergo xenobiotic metabolic processes in the liver. MDA levels were measured using a complex reaction method between MDA and Thiobarbituric Acid (TBA). Measurements were made at a wavelength of 532 nm. The results of making a standard curve obtained by the equation y = 0.0025x + 0.0138 with a value of R2 = 0.9889. The MDA levels can be seen in Table 3 below: The results showed that the administration of 750 mg casein therapy from casein tablets of goat milk yogurt to MDA levels in chicken livers exposed to TCDD for 21 d consecutive could reduce levels of MDA from increasing MDA levels without therapy. The presence of TCDD compounds that are exposed to broilers signifies as a source of free radicals that produce MDA levels as a benchmark for free radicals formed in the body of broilers. In the control liver therapy, there was a decrease in MDA levels from positive control which showed a decrease in MDA levels by 43.74% which was significantly significant (p <0.05) because it had a different notation between positive control and therapy that indicated the treatment results differed greatly or not. as homogeneous. Liver control of broiler chicken therapy obtained MDA levels of 28.54 μg/mL which was lower than the positive control that was equal to 50.74 μg/mL. While the MDA levels in broiler chicken liver increased by 47.16% of the MDA levels of negative control liver that is 26.81 μg/mL lower than the positive controls that significantly affected (p <0.05). Exposure to TCDD increases the level of MDA in the liver. Padaga et.al., (2018) reported the that the antioxidant potential of goat milk yogurt tested against white rats (Rattus norvegicus) as a reprotoxic animal model exposed to TCDD showed a significant effect (p <0.05 ) on serum SOD and MDA levels, testicular MDA and spermatozoa cell density due to exposure to TCDD at doses of 900 mg/kg body weight of goat milk yogurt (Padaga et al., 2018) casein supplementation of goat milk yogurt on dioxin intoxication with rat liver parameters influencing goat milk significantly (p <0.05) to a decrease in the level of the enzyme y-glutamyl transferase (GGT) with an optimal dose of 600 mg/kg/ d. Besides reducing the activity of antioxidant enzymes, such as glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) (Setianingrum et al., 2019) . TCDD compounds induce lipid peroxidation and weaken the antioxidant defense system in various organs (Triebig et al., 1998) .
Effect of Casein Tablet Made from Goat Milk Yogurt on Liver Histopathological Profile in Broiler Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin The liver in humans weighs about 1.5 kg which is the largest exocrine, gland functions as a producer of bile which breaks down fat and endocrine in all metabolic processes, both anabolism and catabolism such as amino acids, fats, and glucoses. Liver cells in protoplasm are nucleated and polyhedral cells that contain many enzymes. TCDD toxicity effects in animals can change the specific morphology that occurs due to hyperplasia and hypertrophy in parenchymal cells resulting in decreased liver function, decreased antioxidant enzyme activity with increased serum and liver damage (Türkez et al., 2013) . Histopathological assessment of the therapeutic effect of 750 mg casein from casein tablets of goat milk yogurt exposed to TCDD in broiler chicken liver tissue was evaluated as in Figures 1, 2, and 3 . Histopathological picture of broiler liver tissue negative control group in Figure 1 (A) contained cells normal hepatocytes (NH), congestion in the central venous lumen (CV), portal vein (PV), and normal biliary tract (BD). (B) there are hepatocyte (H) cells arranged radially from the central vein (CV). Microscopic observations of the chicken liver tissue of the control group showed that the liver lobules were arranged radially from the central vein. The liver lobules appear separated by connective tissue. The portal area consists of a central vein, portal vein, and biliary duct which shows that the hepatocytes appear normal. In contrast to the positive control group that can be seen in Figure 2 below: The results of the histopathological picture of broiler liver tissue in the therapeutic control group showed (A) there was congestion in the central venous lumen (CV). (B) sinusoid dilation (SD) occurs, hemorrhage (H) in sinusoids and hepatocyte pyknosis occurs. Microscopic observation of histopathology in the chicken liver of the therapy group showed that there was congestion of the central venous lumen and sinusoid. Sinusoid shape looks irregular and has moderate dilatation. Some hepatocytes undergo pyknosis. Mononuclear (inflammatory) cells appear to be reduced compared to the positive dick group. Theoretically, the process of liver damage starts from the degeneration process shown by cell swelling. Treatment with TCDD exposure seems to produce an extracellular fluid into large amounts of cytosol. One of the changes caused by toxic chemicals and can cause the formation of free radicals, it is a change in the characteristics of cells and cytoplasmic membranes in cellular components, such as mitochondria and lysosomes that are affected by lipid peroxidation (Karami et al., 2001) . After damaging the cell membrane, toxic chemicals can also reach and destroy the cell nucleus, which causes abnormal cell structure and progresses to necrosis. Necrosis is degradation or irreversible cell disorganization or cell death as a lesion effect, signed by the apparent morphological transformation of the nucleus as pyknosis (solidified cell chromatin), caryoreksis (nucleus rupture) and caryolysis (nucleus disappears) (Ciftci et al., 2013) . Hepatocyte damage and oxidation stress due to TCDD induction have encouraged the development of various studies on active ingredients that are can protect hepatocytes against oxidative stress focused on antioxidants (Turkez and Geyikoglu, 2011) . TCDD given in doses of 10 to 100 ng/kg body weight/day for thirteen weeks to female Sprague Dawly mice can cause liver mutases such as hepatocellular degeneration, inflammation, necrosis, vacolization of the cytoplasm, changes in shape, increased production of superoxide anions, lipids peroxidation, broken DNA stands, and decreased glutathione in brain and liver tissue. Oral administration of TCDD at a dose of 0.1 ng/kg body weight for twenty-seven days in the histopathological description of the liver is hepatocyte vacuolization, inflammation, and increased Kupffer, necrosis, and an increase in the amount of malondialdehyde (MDA) blood plasma (Hassoun et al. 2000) .
CONCLUSION Therapy with 750 mg Casein tablets made from goat milk yogurt can prevent the increase of TCDD residual levels, malondialdehyde (MDA) levels and can prevent damage to the histopathological picture of broiler chicken liver tissue exposed to 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD). So that it can be used as an antioxidant nutraceutical. Figure 1 . 1 Figure 1. Results of Hematoxylin-Eosin Staining of Broiler Chicken Liver with 200× and 400× Magnification. (A.B) Negative Control Group.
Figure 2 . 2 Figure 2. Results of Hematoxylin-Eosin Staining of Broiler Chicken Liver With 200× and 400× Magnification. (A.B). Positive Control Group.
Figure 3 . 3 Figure 3. Results of Hematoxylin-Eosin Staining of Broiler Chicken Liver With 200× and 400× Magnification. (A.B). Therapeutic Control Group.
: Table 1. Composition of Casein Tablets Component Concentration Casein Goat Milk Yogurt 250 mg Collidon 30 600 mg Collidon Cl 350 mg Lactose 9300 mg Aerosil 200 mg Magnesium Stearic 200 mg Gelatin 5%
Table 2 . 2 Average TCDD Residue Levels of Broiler Liver Treated with 750 mg Casein Tablet % increase in levels of TCDD residues in the negative control of broilers obtained by comparison of the average levels of TCDD residues in positive control of broiler chickens. % decrease in TCDD residue levels in control of broiler chicken therapy obtained by comparison of the average levels of TCDD residue in positive control of broiler chickens. The different notation indicates a significantly different effect (p <0.05). Made from Goat Milk Yogurt Treatment Group Residue Levels TCDD Increased TCDD Decreased in TCDD (mg/L) (Mean±SD) Residue Levels (%)* Residue Levels (%) Negative Control 0.1039±0.0102 a - - Positive Control 0.1407±0.0224 b 26.15 - Therapeutic Group 0.1326±0.0077 b - 5.75 Note: *
Table 3 . 3 Average MDA Levels of Broiler Liver Treated with 750 mg Casein Tablet from Made % increase in MDA levels in the negative control of broiler chickens obtained a comparison of the average MDA levels in positive control of broiler chickens. % decrease in MDA levels in control of broiler chicken therapy obtained by comparison of the average MDA levels of positive control of broiler chickens. The different notation indicates a significantly different effect (p <0.05). Goat Milk Yogurt Treatment Group MDA Levels (μg/mL) Increased MDA Decreased MDA (Mean±SD) Levels (%)* Levels (%) Negative Control 26.8133±12.1363 a - - Positive Control 50.7467±3.2166 b 47.162 - Therapeutic Group 28.5467±8.6511 a - 43.746 Note: *
|
10.1038/s41598-017-14388-8
|
cc-by
| null | null |
openalex
|
Hybrid organometallic systems offer a wide range of functionalities, including magnetoelectric (ME) interactions. However, the ability to design on-demand ME coupling remains challenging despite a variety of host-guest configurations and ME phases coexistence possibilities. Here, we report the effect of metal-ion substitution on the magnetic and electric properties in the paramagnetic ferroelectric NH 2 (CH 3 ) 2 Al 1-x Cr x (SO 4 ) 2 × 6H 2 O. Doing so we are able to induce and even tune a sign of the ME interactions, in the paramagnetic ferroelectric (FE) state. Both studied samples with x = 0.065 and x = 0.2 become paramagnetic, contrary to the initial diamagnetic compound. Due to the isomorphous substitution with Cr the ferroelectric phase transition temperature (T c ) increases nonlinearly, with the shift being larger for the 6.5% of Cr. A magnetic field applied along the polar c axis increases ferroelectricity for the x = 0.065 sample and shifts T c to higher values, while inverse effects are observed for x = 0.2. The ME coupling coefficient α ME = 1.7 ns/m found for a crystal with Cr content of x = 0.2 is among the highest reported up to now. The observed sign change of α ME with a small change in Cr content paves the way for ME coupling engineering.Realization of the effective interactions between magnetic moments and electric charges constitutes an important task for modern solid state physics 1, 2 and spin electronics . The principal motivation is to establish electric control of magnetism for low-power spintronic structures. For this reason large values of magnetization and electric polarization are often motivating factors for multiferroic materials research . Although large magnetization is expected from ferromagnetic ordering, ferromagnetism and ferroelectricity tend to be mutually exclusive in a single phase 10 and the largest magnetoelectric coupling is mostly seen in antiferromagnets at symmetry-breaking spin reorientation transitions 11 . However, ME coupling of higher orders can be symmetry independent and exists for other types of magnetic orderings 12, 13 . In particular, a large ME effect was reported to exist in the paramagnetic [(CH 3 ) 2 NH 2 ]Mn(HCOO) 3 14 . Along with other successful examples , this result demonstrates a large potential of organic-inorganic materials 21 in the research of ME compounds and beyond 22, 23 . Because electric order in the lattice is more fragile than a magnetic one, a promising strategy to achieve their safe coexistence can be implementation of magnetic interactions into already known electrically polar compounds rather than vice versa. In this respect the organic-inorganic hybrid frameworks offer indeed an abundance of possibilities . Here we study the ferroelectric NH 2 (CH 3 ) 2 Al(SO 4 ) 2 × 6H 2 O (DMAAS) crystal which belong to organic-inorganic functional materials known to be electrically polar below 152K 33, 34 . The crystal structure of DMAAS is built up of Al cations coordinated by six water molecules (i.e. water octahedra), regular (SO 4 ) 2-tetrahedra and [NH 2 (CH 3 ) 2 ]+ (DMA) cations, all hydrogen bonded to a three dimensional framework (Fig. 1 ). Thus, in the crystal structures of [NH 2 (CH 3 ) 2 ]Al 1-x Cr x (SO 4 ) 2 × 6H 2 O with x = 0.2 and 0.065 octahedra are occupied by statistical mixtures of magnetic Cr -and nonmagnetic Al -atoms. With cooling this crystal exhibits a second order phase transition at T c = 152 K from paraelectric but ferroelastic (T > T c ) to ferroelectric (T < T c ) phases. The phase transition is of the order-disorder type with a symmetry change 2/m → m. It is connected with ordering of the polar DMA cations which execute hindered rotations around their C-C direction in the paraelectric phase and order only in the spatio-temporal average in the ferroelectric phase 35 . Metal ion isomorphous substitution in the above mentioned family of compounds can be an additional degree of freedom in the composition-property engineering 36, 37 . Here we report that the incorporated magnetic Cr cations can participate in ME interactions in the [NH 2 (CH 3 ) 2 ]Al 1-x Cr x (SO 4 ) 2 × 6H 2 O crystals with ability to tune magneto-electric functionality.
Results Initial DMAAS crystals usually grow in a monodomain ferroelastic state. The ferroelastic domains may nevertheless appear during polishing or other type of mechanical treating. However, if Cr is introduced this tendency is inversed: the samples predominately grow in a polydomain ferroelastic state (Fig. 2 ). In this case the EDX analysis of the neighboring domains shows a different content of chromium. The quantity of Cr in oppositely stressed domains is equal to 17.6% and 20% respectively. This rule, however, does not apply to minor quantity of samples yet grown in the single domain state where chromium is distributed evenly within a sample. It is observed that thermodynamical conditions of the growth are highly stress-dependent and allow variation of the Cr distribution within the sample volume. This intriguing result, however, deserves separate investigations that are beyond the scope of this paper. During our further discussion we will consider only the crystals grown in a polydomain states as this type of growth is statistically overwhelming. The average thickness of domains was found to be equal to 100-120 μm, with the optical indicatrix disorientation angle of 2θ = 40. The existence of a polydomain ferroelastic state was also verified by scanning electron microscopy imaging (Fig. 2c, d ). Similarly to the initial compound our samples show large peaks in the dielectric permittivity due to the polar state formation at T c = 153 K which is characteristic for proper ferroelectric phase transitions (Fig. 3 ). Notably, the value of the dielectric permittivity is almost three orders of magnitude larger for an AC electric field applied along the c axis confirming the spontaneous polarization direction (Fig. 3a ). The temperature A distinct clear peak in the current is observed at the same temperature where both dielectric permittivity and thermal expansion also show anomalies. A DC electric field of 7.9 kV/m applied during cooling reveals peaks in the pyroelectric currents for both samples and demonstrates Cr dependent T c evolution. The temperature dependences of the dielectric properties for both investigated crystals fairly well correlate with the data of previous investigations 37 and obey the Curie-Weiss law both in the paraelectric (+) and ferroelectric (-) phases in the vicinity of the ferroelectric phase transition. Together with the data of DSC study 37 this clearly confirms a second order of the phase transition. No other anomalies were observed in the pyro-current temperature dependences at cooling of both samples down to 1.6 K. Therefore, one can conclude that the ferroelectric phase exists in our DMAAl 1-x Cr x S crystals in the temperature range from T c down to 1.6 K. This conclusion is also confirmed by the temperature dependences of the electric polarization (Fig. 4a ) measured after ferroelectric saturation occurring above 250 kV/m 38 . The magnetic susceptibilities of DMAAl 1-x Cr x S complexes are depicted in the inset to Fig. 4b along with electric polarization data (Fig. 4a ). The pure complex without Cr (x = 0) is diamagnetic in the whole studied temperature range with residual susceptibility χ 0 given in Table 1 . Isomorphous substitution of Al with Cr leads to the appearance of a paramagnetic fraction below 100 K for x = 0.06 and to a paramagnetic behaviour and, thus positive χ(T) for x = 0.2 (inset to Fig. 4b ). Both these susceptibilities fit excellently to modified Curie-Weiss (CW) law (χ = C/T + χ 0 ) in the temperature range 50-300 K (inset to Fig. 4b ). As one can see from Table 1 the χ 0 values obtained from the fit agree well with those of initial DMAAS crystal. This confirms that Cr-atoms are embedded into a diamagnetic matrix. Effective magnetic moments μ eff deduced from the fit are close, indicating no Cr-Cr interactions in the studied compounds. As it is known Cr 3+ usually forms octahedral complexes. The Al 3+ ions (in this crystallographic position the Cr substitution is expected) center octahedral voids in the studied structures (Fig. 1 ). Both these facts hint toward +3 oxidation state for Cr (i.e. 3d 3 electronic configuration) and thus, a low spin type of complex in agreement with earlier studies 36 . Interestingly, no anomalies are seen in the magnetic susceptibilities at ferroelectric T c as it would be expected from ref. 14 . However, careful examination of the derivatives dχ/dT reveals a clear deviation from linearity near T c (Fig. 4b ) for the DMAAl 1-x Cr x S crystals and temperature independent linear behavior for the initial sample. Even more, the magnetic susceptibility derivative for the crystal with x = 0.065 shows an upturn towards low temperatures while for x = 0.2 an upturn occurs towards high temperatures. Such a different behavior correlates well with the different magnetic field dependence of the pyroelectric current magnitude and its temperature position for both samples (Fig. 5 ). With increasing of magnetic field the intensity of the peak in pyro-current increases and shifts towards higher temperatures (promotes ferroelectricity) for the compound with x = 0.065, while for x = 0.2 the opposite effects are observed (Fig. 5 ). The ME coupling coefficient α ME in the units of [s/m] is defined here as: ∫ α = - = H I I dt 1 ( ) ME H O H where I is the pyroelectric current density and H is the magnetic field applied (Hμ 0 = 6 T) during the measurements (Fig. 5 ). As one can see from the ME coupling coefficient presented in Fig. 6 , the effect of coupling is stronger for the crystal with Cr content x = 0.2. As expected the absolute value of α ME decreases monotonically till the FE Curie temperature is reached. However, both crystals also show a discontinuity in ME coupling near T C (inset to Fig. 6 ) implying the existence of a small coupling even in the paraelectric and paramagnetic region. The fact that the ME coupling coefficient changes its sign as a function of Cr content as well as the large level of the coupling itself points towards the possibility to tune the ME response in such compounds. The ME properties can be tentatively explained by the twofold effect. Firstly, the introduction of larger (Ri Cr = 0.6115 Å; Ri AL = 0.535 Å) 39 and magnetic Cr generates strains and secondly, makes the compound more sensitive to magnetic field. The magnetoelectric coupling here can arise via stress mediated contribution. With Cr content increase the overall sample deformation goes via critical point modifying local magnetism and polarization. This assumption seems to be in agreement with the sensitive ferroelastic structure. This issue, however, deserves a separate study including optimal Cr content determination. In conclusion, this study reports the successful creation of paramagnetic order in the initially diamagnetic DMAAS crystal by isomorphous substitution of metal ion. Such action intimately connects apparently separated magnetic and electric subsystems, as evidenced by the temperature dependence of the derivative magnetic susceptibility. We have successfully generated a large ME coupling and importantly demonstrated the possibility to tune its sign, depending on the Cr content. Moreover, our results suggest that ME coupling can exist in the paramagnetic compounds without easily noticeable magnetic anomalies near FE transitions, and special care should be taken for such evidence. From the ferroelectric point of view, we show that partial isomorphous substitution with Cr metal ions leads to an anoticeable shift of the phase transition and can be used to increase FE polarization and T c . In particular, in comparison with initial DMAAS crystal, the phase transition temperature T c in the crystal doped with Cr 3+ (6.5%) is shifted toward higher temperatures by 2.6 K, whereas for a higher chromium concentration (20%), this shift is diminished to 0.6 K. The Cr distribution in such samples can also be controlled by stress-assisted material growth conditions and provides additional degrees of freedom for organometallic materials engineering. Our study motivates further investigations in the area of paramagnetic organic-inorganic materials, with the design of ME interactions at room temperature as the next milestone.
Methods Single crystals of [NH 2 (CH 3 ) 2 ]Al 1-x Cr x (SO 4 ) 2 × 6H 2 O (DMAAl 1-x Cr x S) were grown from an aqueous solution containing the metal sulphates in a stoichiometric ratio and dimethylammonium sulfate at a constant temperature of 303 K by slow evaporation method. The molar ratio of Al 3+ : Cr 3+ in the solution was equal to 1: 0.065 and 1: 0.2, respectively. This ratio in the samples was controlled by SEM using a REММA-102-02 (SELMI, Ukraine) scanning electron microscope. Quantitative electron probe microanalysis (EPMA) of the phases was carried out using an energy-dispersive X-ray (EDX) analyzer with the pure elements as standards (the acceleration voltage was 20 kV; K-and L-lines were used). The obtained values of Al 3+ : Cr 3+ molar ratio were found to be 0.065 ± 0.006 and 0.2 ± 0.02 (for a single domain samples) and correspond to those in the reacting solution. The surface morphology was studied by SEM. The scanning of sample surface was performed by an electron beam with energy of 15 and 20 kV and a diameter of 5 nm in the secondary electron image regime. To prevent charging during SEM cycling, the sample was covered by a thin graphite layer transparent for the electron beam. The thermal expansion was measured using a home-built capacitive dilatometer. The measurements of the real part of dielectric permittivity and conductivity were carried out using the traditional method of capacitor capacitance measurement. The capacitance was measured using an automated setup based on a LCR-meter HIOKI 3522-50 LCF HiTester. The spontaneous polarization measured using Keithley 6517 A electrometer. The magnetic susceptibility was measured using a commercial magnetometer Quantum Design MPMS-3 in the temperature range 1.8-300 K and magnetic fields up to μ 0 H = 7 T. For both polarization and magnetic measurements electric and magnetic fields were applied perpendicular to the crystallographic plane in the monoclinic crystal structure (parallel to the polar axis). Figure 1 . 1 Figure 1. Crystal structure of non-centrosymmetric monoclinic [NH 2 (CH 3 ) 2 ]Al(SO 4 ) 2 × 6H 2 O at 135 K. The Al atoms are in the centers of yellow [H 2 O] 6 -octahedra. In the case of Cr substitution the octahedra are occupied by statistical mixtures of magnetic Cr and nonmagnetic Al atoms. The [SO 4 ] 2-tetrahedra are depicted in grey color.
Figure 2 . 2 Figure 2. The ferroelastic domain structure at 300 K. Polarization microscopy pictures on the cut of DMAAl 0.8 Cr 0.2 S crystal perpendicular to (a,b) and corresponding view of the (001) (c) and (310) (d) surfaces of the same crystals obtained using a scanning electron microscopy in СОМРО and TOPO regimes respectively.
Figure 3 . 3 Figure 3. Temperature dependence of the structurally correlated electric properties. (a) The real part of the dielectric permittivity ε' a and ε' c for DMAAl 0.8 Cr 0.2 S crystals. (b) The thermal expansion measured along the principal cuts of DMAAl 0.8 Cr 0.2 S crystals. (c) Pyroelectric currents for samples with x = 0.2 and 0.065 Cr content.
Figure 4 . 4 Figure 4. Temperature dependence of magnetic and electric properties. (a) Electric polarization. (b) The derivatives of susceptibilities for DMAAl 1-x Cr x S crystals with different Cr content. Insets to figures (a) and (b)show respectively a variation of the ferroelectric temperature T c and magnetic susceptibilities from which magnetic parameters are determined via CW fit (Table1).
Figure 5 . 5 Figure 5. Magnetic field influence on the pyroelectricity. Upper panel: the sample with the Cr content of x = 0.065; Lower panel: the sample with the Cr content of x = 0.2. An opposite behavior in both magnitude and temperature position of the pyroelectric peak is observed.
Figure 6 . 6 Figure 6. Temperature dependence of the ME coupling coefficients for parallel orientation of magnetic and electric fields. Inset shows a zoomed region near T c .
Table
-content, x χ 0 (10 -4 emu mol -1 ) μ eff (μ B ) ). Cr0 -2.26(7) 0.06 -2.40(5) 1.47(1) 0.2 -2.49(8) 1.91(1)
Table 1 . 1 Magnetic parameters for DMAAl 1-x Cr x S crystals.
SCIENTIFIC REPORTS | 7: 14109 | DOI:10.1038/s41598-017-14388-8
|
10.17511/ijoso.2018.i03.05
|
cc-by
| null | null |
openalex
|
Background: Liver is the most frequently injured solid intra abdominal organ in abdominal trauma. Exsanguination is the main cause of death due to liver trauma. Although non-operative management of hepatic trauma has been utilized with increasing frequency a significant percentage of unstable liver trauma still require operative treatment. The aim of the present study was to examine the results of the operative treatment of patients with unstable liver trauma and prevent prolonged hospital stay. Patients admitted following Liver Trauma in the department of General Surgery, MOSC medical college Hospital, Kolenchery duringthe two year period from January 2013 to January 2015 were included in the study. Methods: This was a retrospective study of patients with Hepatic trauma admitted to the department of General Surgery, MOSC Medical College Hospital, Kolenchery, Ernakulam from January 2013 to January 2015. The diagnosis of Liver trauma was made pre-operatively with imageology and assessed clinically prior to surgery. Severity of liver injury was graded, treatment details collected and factors contribution to prolonged hospital stay were noted. These patients were later followed up for a period of one year. Results: During the two year period, 105 patients who were diagnosed to have liver injury were included in the study. Age varied from 12-75 years. Males outnumbered females (88.5% vs. 11. 43 %). 93 patients were with blunt abdominal trauma (88.5%). 66(62.85%) patients were in shock when they presented to the ER. 36 (37.15%) patients were haemo-dynamically stable. 57 patients (54.28%) had associated injuries like multiple rib fractures and splenic injuries. Exploratory laparotomy and control of bleeding, hepatorapphy and local debridement was done. Nine patientsrequired relaparotomy and omental packing. Fifteen patients succumbed to liver injury. The postoperative period was delayed in those patients who had other visceral injury. Follow up of cases for a period of 1 year was done and there was no late complication like intra-abdominal abscess, coagulopathy, bile leak or hepatic abscess. Conclusion: Emergency laparotomy with hemostasis and repair liver injury in unstable cases and select stable cases savestime and life of the patient, it is cost effective and hospital stay and systemic complications are minimalIntroduction The Liver is the most commonly injured intraabdominal solid organ in both blunt and penetrating trauma because of its size and location . Exsanguination is the major cause of death in hepatic trauma with a (mortality [S1] of 10-15 . Liver trauma should be considered in all patients with penetrating or blunt trauma, particularly in hypotensive patients with penetrating or blunt trauma on the right side . Blunt trauma more commonly affects the right lobe of liver, particularly the posterior sector . Although conservative treatment of low grade liver injuries is practiced Manuscript Received: 10 th September 2018 Reviewed: 20 th September 2018 Author Corrected: 24 th September 2018 Accepted for Publication: 28 th September 2018 nowadays emergency laparotomy and hemostasis with repair of liver injury has a definite role in unstable cases and reduces the mortality and morbidity to a great extent . The criteria for conservative treatment of liver injuries includes Hemodynamic stability, normal mental status and absence of clear indications for laparotomy such as peritoneal signs, low grade injuries (grade1 to 3) and transfusion requirements less than 2 units of blood . Most series report a success rate of almost 90% for conservative treatment of liver injuries. The success rate is 95% for low grade liver injuries 1 to 3 which falls to 75% for grade 1V to VI injuries . Conservative
Original Research Article
Surgical Update: International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 122 | P a g e management has shown a lower rate of complication (0-11% . However a significant number of liver trauma cases especially the grade 3 to grade 5 cases benefit by timely and early laparotomy, hemostasis and repair of liver injury especially in a rural hospital where prolonged critical care is not always possible. Angiography and selective embolization is increasingly used in the treatment of persistent bleeding cases and this may result in more cases being treated conservatively . However these modalities may not always be available. The principles of operative treatment are the same for all cases of liver injuries. They include damage control surgery that includes arrest of bleeding, removal of devitalized tissueand prevention of biliary complications in unstable patients . Most of the liver injuries can be managed with simple procedures like suturing, debridementor packing with omentum gel foam. The mortality and morbidity associated with liver injuries varies drastically from 1.5% to 31%. Mortality is low with penetrating injuries whereas the mortality associated with blunt trauma is as high as 31% depending on the mechanism of injury. Early surgical intervention reduces mortality and morbidity and saves patients life, time and money.
Material and Methods Study Setting: The study was conducted in department of General Surgery MOSC Medical College, Kolenchery.
Study Design: Retrospective observational study Study Period: Between January 2013 and February 2015.
Sample Size: 105 Cases of blunt or penetrating abdominal injury with preoperative ultra sound or CT scan diagnosis of liver injury Exclusion:-We did not include patients who died at the scene or on their way to hospital. Patients who arrived in the Emergency department in a state of cardiorespiratory arrest and whose period of attempted resuscitation did not exceed 15 minutes were also excluded from the study.
Ethical Consideration: The study was approved by the institutional human ethics committee. Informed written consent was obtained from all the study participants. Study procedure-Patients fulfilling the inclusion criteria (105 cases) were identified and studied who presented to our emergency department with liver injury. Patients were resuscitated in the Emergency department and optimized. Diagnosis was established by either by surgery, organ imaging by computerized tomography or ultrasound. Based on the imaging and clinical findings that included vital signs patients were divided into stable and unstable cases and classified (according [S1] to the liver injury scale (1994 revision of the American Association for the Surgery of Traumaliver injury scale), the most widely used liver injury grading system used at the time of study . Unstable cases were taken up for laparotomy where as the stable cases were initially treated conservatively and those cases which showed clinical deterioration during the follow up were taken up surgery. Emergency exploratory laparotomy was done through a midline vertical incision and hemostasis was attempted with Pringles maneuver, Gauze packing, simple ligation, hepatorapphy with catgut and with omental packing and debridementin those cases where there was uncontroll able hemorrhage. Postoperative follow up was like in any other case of emergency laparotomy but with specific emphasis on vital signs, clinical improvement or deterioration of the patient.
Results During the study period 105 patients who presented to emergency department with liver injury were (90 males [85.71%] and 15 females [14.29%]). The majority of patients 93 (88.57%) sustained blunt trauma and 12 had penetrating injury (11.43%). Patients belonged to the wide age group from12 years to 71 years. Maximum incidence was in the 21-30 age group (28.7%) followed by 10-20 age group (25.71%). 72(68.57%) patients were brought to the ER following road traffic accident, 21(20%) patients were admitted with a history of fall from height. and 12 (11.4%) patients were admitted following stab injury. Associated Injuries-The associated injuries were as follows.
Original Research Article Patients with multiple rib fractures-24 patients (22.85%) Haemothorax-18 patients (17.42%) splenic injury-3 patients multiple mesenteric tear-12 patients head injury10 patients long bone fracture 16 patients Among these multiple rib fractures, head injury and long bone fractures influenced the post-operative recovery and were significant determinants of successful recovery. However the major determinants were hemorrhage and shock. Surgery-All 69 unstable patients including three patients in grade 2 liver injury who were initially stable at presentation were optimized prior to exploratory laparotomy. Abdomen was entered through midline vertical incision. Hemorrhage was controlled by digital pressure, suture ligation, diathermy, omental packingand lobectomy depending on the intraoperative findings. Re-laparotomy was done in 9 patients.Rebleeding in three patient which was controlled by catgut suturing, omental packing in one patient and Segmentectomy was done in the other two. Three patients were operated for perihepatic packing removal done as part of damage control surgery and the rest three patient required relaparotomy for drainage of hematoma and perihepatic collection since radiological intervention was successful. During the postoperative period fifteen patients deteriorated and they couldnot be revived in spite of resuscitation. The cause of death in one patient was Grade v liver injury with massive bleed and cardiac arrest during the immediate postoperative period. The second patient had Grade 3 liver injury with splenic injury and diaphragmatic hernia who died on the 5th postoperative period following pneumonia and sepsis. Three patients died because of massive rebleed during the immediate postoperative period. Four patients died due to associated head injury. Five patients due to sepsis and renal dysfunction. And one patient developed myocardial infarction in post op period. The mortality rate in our study was 14.28%. Various studies have reported mortality rates ranging from 18% to 36%. Fifteen patients developed sepsis out of which ten survived. Wound infection was noted in twenty-three patients. Blood transfusion was a major concern and 2 to 50 units of blood was transfused. FFP and platelets were transfused during the postoperative period. Transfusion requirements were judged by perioperative blood loss, serial blood investigations and clinical judgment. Patients other than those with associated injuries like head injury, long bone fracture had earlier discharge. The mortality rate in our study was 14.70% which was much less than the rate reported in literature. The unstable cases were taken up for laparotomy and were treated by suture ligation, digital pressure, diathermy coagulation, omental packing and debridement. All the procedures were equally good. The survival rate was (85.7%) with (early [S1] recovery, short hospital stay, and minimal complications. In our study we found that early operative intervention in unstable cases in spite of being low grade injuries results in early recovery with very few complications. Regular postoperative follow up was done for a period of 1 year and all the patients were asymptomatic as proved by the laboratory and imageology investigations.
Original Research Article
Surgical Update: International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 124 | P a g e
Discussion Liver is the commonest intraabdominal solid organ to be injured in blunt orpenetrating trauma because of its size, location and relative fragile parenchyma . Severe hepatic trauma is a major cause of death in abdominal trauma. With the developments in imageology most hepatic injuries can be treated conservatively. The criteria for non operative treatment of hepatic injuries includes hemodynamic stability, absence of peritonitis, low grade hepatic injuries and transfusion requirements of less than 2 units of blood . Most series show a success rate of 90% for conservative treatment . However a small but significant percentage of unstable patients with liver injury benefit from early and timely surgical intervention resulting in improved quality of life to the patient. Most hepatic injuries were caused by blunt trauma occurring during motor vehicle accidents which is as per literature . Most hepatic injuries were associated with other visceral injuries 60/105 (57.1%) as reported other literature . Mortality in low grade hepatic injuries (grade 1 to 111) is almost always caused by associated injuries and not by liver injury as describes in literature (10.4%). In our study the mortality in low grade liver injuries was 12/96 (12.5%). However mortality associated with high grade injuries (grades IV to VI) varies from 18% to 36% (11) . In our study the mortality in high grade liver injury was 3/9 (33.3%). Presence of shock at the time of admission is associated with higher mortality . The aim of treatment while dealingwith an unstable liver injury was to control bleeding as quickly as possible and thus limit the extent of liver injury. Direct suturing of the bleeding artery,suture approximation of the liver wound edge (hepatorapphy), hepatic artery ligation, omental packing, resectional debridement, anatomic hepatic resection, perihepatic packing are the procedures commonly employed . In our study majority of the bleeding was controlled with simple suturing and diathermy coagulation. When active bleeding is encountered on
Conclusion Based on our findings and study we found that for unstable cases of liver injury irrespective of the grade of liver injury timely surgical intervention has a role in saving lives, reducing hospital stay and relatively uneventful post-operative period and follow up. It was found that emergency laparotomy and control of bleeding and repair of liver injury by simple methods was found to be very affective and improve the quality of the patient. Associated visceral injuries contributed to the mortality and morbidity of the patient. According to the literature althoughconservative treatment of liver injury is recommended for grade 1, grade 2 and select grade3 caseswe found that early intervention irrespective of grade of injury in unstable cases improved the quality of life of patient.
Contribution by authors: Dr Satish G Prabhuguided the study and was instrumental in preparing the manuscript. Dr George Abraham edited the manuscript and added the requisite inputs as and when required, Dr Jayant B N compiled the cases and prepared the draft. Surgical Update: International Journal of Surgery & Orthopedics Available online at : www.surgicalreview.in 123 | P a g e
Table - 1: Grades of Injury & condition of patients. Grade of liver injury Stable Unstable - According to table 1, in our study majority of the cases were unstable cases 66/105 (62.8 %). The majority of the cases were grade 2 liver injury 54/105(51.4%). Unstable cases with grade 2 liver injury accounted for 39/68 (57.3%) cases. i 9 6 ii 15 39 iii 15 12 iv 0 6 v 0 3 vi 0 0 Total 39 66
The cause of mortality in low grade injuries (1 to 3) is associated injuries while in high grade injuries (4 to 6) the liver injury itself resulting in exsanguinations the cause of death liver tissue with concomitant suture ligation of the bleeding vessels is done . Omental packing deep in the liver with reinforcing sutures is a useful procedure. Omentum provides an excellent source of macrophages and it fills a potential dead space with viable tissue . In our study we had done omental packing in one patient. Anatomical resection of the liver is seldom done and it is replaced with resectional debridement. In a large series of 5000 cases of hepatic trauma hepatic resection was done only in 7.5% patients and the mortality were as high as 52% . Perihepatic packing is done by keeping roller guaze around the liver and is very useful in patients with other intra-abdominal injuries and shock. Re exploration for pack removal was done after 48 to 72 hours. The association of hepatic abscess as per literature was 29% . However in our study there was no reported case of hepatic abscess development during the follow period. The most prevalent complication is wound infection. It was more commonly seen in those with poly trauma. table inflow occlusion of the liver should be performed by compressing the hepato-duodenal ligament with a vascular clamp (Pringles maneuver). When bleeding continues it is from the hepatic veins or the IVC. Direct suturing of the arteries isrecommended when the bleeding is from the branches of the hepatic arteries or tributaries of the portal veins . When a segment of the liver is damaged, debridement of the devitalized
|
10.7759/cureus.996
|
cc-by
| null | null |
openalex
|
Both polycystic liver disease (PLD) and sarcoidosis can involve liver. Most of the time, liver disease in both conditions is asymptomatic, but they can rarely cause portal hypertension. Our aim is to report a case of a 51-year-old female with a history of adult dominant polycystic kidney disease (ADPKD) and sarcoidosis who presented with multiple episodes of hematemesis. An endoscopy showed grade 3 esophageal varices. A computed tomography (CT) scan of the abdomen showed ascites with polycystic liver, nodular contour, and calcified granuloma. PLD can cause portal hypertension due to fibrosis or large cysts compressing on the portal vein. On the other hand, sarcoidosis causes portal hypertension by formation of arteriovenous (AV) shunts or fibrosis in areas of granulomas. Both conditions are diagnosed on imaging. There is no approved medical treatment for PLD; the only curative treatment is liver transplantation. Asymptomatic hepatic sarcoidosis does not need any treatment. The recommended treatment is corticosteroids for both isolated and systemic sarcoidosis. ADPKD and sarcoidosis can involve multiple organs. The presence of both conditions can accelerate the disease process and could be a therapeutic challenge. Early abdominal imaging during the course of both diseases can improve the outcome by decreasing the diagnostic window.Introduction Polycystic liver disease (PLD) is one of the rare conditions with a prevalence of 1/100,000 to 1/1000,000. Although typically asymptomatic, PLD can complicate the hemorrhage due to rupture of cysts, infection, and portal hypertension and, rarely, end stage liver disease . There are very few cases in literature where polycystic liver disease presented with complication of portal hypertension. Sarcoidosis is a systemic granulomatous disease; it can involve the liver, but in most of the cases patients are asymptomatic. Cirrhosis and portal hypertension develop in less than one percent cases of sarcoidosis . We present a case where adult dominant polycystic kidney disease (ADPKD) and sarcoidosis coexisted and presented as decompensated liver disease.
Case Presentation A 51-year-old female presented to the emergency department after several episodes of hematemesis and severe epigastric pain. She had a past medical history of pulmonary sarcoidosis, ADPKD requiring bilateral nephrectomy and left kidney transplant, and is currently on immunosuppressive therapy. On admission, her vitals were stable. Her hemoglobin was 9.8 g/dl. Her total bilirubin was 0.2 mg/dl, alkaline phosphates 74 U/L, aspartate aminotransferase 24 U/L, alanine aminotransferase 20 U/L, blood urea nitrogen 49 mg/dl, and creatinine 1.07 mg/dl. Electrolytes were normal. She underwent emergent esophagogastroduodenoscopy (EGD), which showed grade 3 esophageal varices and four of them were banded. Octreotide and pantoprazole drip were administered. A computed tomography (CT) scan of the abdomen showed ascites with polycystic liver, nodular contour, and several sites of calcifications (most likely calcified granulomas) (Figure 1 ). An abdominal ultrasound showed a heterogeneous liver with mild contour and patent portal circulation. A previous magnetic resonance imaging (MRI) of the pelvis, which was done two years ago, showed similar small cysts in the parenchyma; however, the calcifications and nodularity of the liver were new. Diagnostic paracentesis revealed serum to ascitic albumin gradient (SAAG) of 1.9 consistent with portal hypertension. The patient was started on non-selective beta blocker for stabilization of varices. Ceftriaxone was given for prophylaxis for spontaneous bacterial peritonitis. The patient's bleeding was controlled, and the hemoglobin level was stable during the hospital stay. She refused further workup including liver biopsy.
Discussion Polycystic liver disease occurs either in isolation or as part of ADPKD or uncommonly adult dominant polycystic liver disease (ADPLD). Polycystic liver disease involves mutation of polycystin 1, polycystin 2, hepatocystin, and Sec63, which leads to the failure of intralobular bile ducts to involute during embryonic development. The risk factors for hepatic cyst growth involve age, female gender, and size of renal cysts . Hepatic cystic disease is usually asymptomatic and does not need intervention; rarely, extensive cystic disease may lead to liver failure. There are few cases in literature where polycystic liver disease complicated into portal hypertension either with fibrosis or with extensive cystic disease . The pathogenesis includes two processes. First, polycystic disease decreases hepatic vein flow, and second cysts can compress the portal vein leading to portal hypertension . Sarcoidosis is a systemic granulomatous disease that can commonly involve the liver. Hepatic sarcoidosis is usually asymptomatic, and rarely can it progress to cirrhosis, portal hypertension, cholestasis, and Budd-Chiari syndrome . The pathophysiology of portal hypertension in sarcoidosis is unknown. Formation of AV shunts in areas of granulomas could be a cause; AV shunting increases the resistance which leads to portal hypertension. Another suggestion is that healing fibrosis in areas of granuloma increases the resistance, which can lead to portal hypertension with cirrhosis. Another theory suggests that ischemic changes caused by granulomatous phlebitis of portal and hepatic veins can lead to cirrhosis and portal hypertension . Both conditions are diagnosed on imaging. Abdominal ultrasound and CT scan are the first line modalities. Liver biopsy supports diagnosis of sarcoidosis and rules out other conditions. Liver function abnormalities are common in symptomatic patients [3, 6] . The presence of both sarcoidosis and polycystic liver disease increases the risk of development of decompensated liver disease or liver failure as compared to single disease. To our knowledge there is no case in literature where both conditions involved the liver simultaneously. There is no approved medical treatment for PLD. Studies have shown somatostatin analogues and mammalian target of rapamycin (mTOR) inhibitors efficacy as compared to placebo. Radiological treatment includes aspiration followed by sclerotherapy for large cysts. Surgical treatment involves fenestration, segmental hepatic resection, and liver transplantation. Liver transplantation is the only curative treatment for PLD . The Model of End Stage Liver Disease (MELD) score is commonly used for liver allocation. Usually liver functions are preserved in PLD, and the MELD score is not the best criteria to assess for liver transplantation. Some studies suggested the use of the MELD priority score to improve the outcome . Asymptomatic hepatic sarcoidosis patients do not need any treatment. The recommended treatment is corticosteroid for both isolated and systemic sarcoidosis. Corticosteroids improve liver function tests but do not improve progression of disease. The survival in patients who have undergone a liver transplant with hepatic sarcoidosis is comparable to other liver diseases leading to transplant .
Conclusions Polycystic liver disease and sarcoidosis are rare etiologies for portal hypertension. The presence of both conditions can accelerate the disease process and could be a therapeutic challenge. An early abdominal imaging during the course of a disease can improve the outcome. FIGURE 1 : 1 FIGURE 1: Abdominal CT scan showing multiple cystic hypodensities with calcification in liver parenchyma.
Amjad et al. Cureus 9(1): e996. DOI 10.7759/cureus.996
|
10.3389/fphys.2019.00358
|
cc-by
| null | null |
openalex
|
This Research Topic provides an updated view of rotary F-and V-ATPases, including aspects of their complex chemo-mechanical coupling and of their role in physiology and pathology. F-and V-ATPases are unique and ubiquitous rotary machines that evolved from a common ancestor and share the ability to pump H + or Na + powered by the energy deriving from ATP hydrolysis. The F-types (F-ATP synthases) also catalyze the synthesis of ATP using the electrochemical gradient of (usually) protons generated by respiration, providing most cellular ATP. Both F-and V-ATPases consist of a hydrophilic globular catalytic domain (F1 or V1, respectively) composed of an asymmetric hexameric ring with a central stalk located inside the ring; a hydrophobic membrane-embedded domain (Fo or Vo, respectively) which allows ion translocation across the membrane, and a peripheral domain with a stator function composed by one (in F-types) or three (in V-types) stalks. In their fascinating review article, Singharoy et al. discuss a number of recently published crystal structures of the V1 sub-complex from Enterococcus hirae V-ATPase. This V-ATPase functions as a primary Na + pump maintaining ion homeostasis and providing high salt tolerance to this organism The snapshots corresponding to different catalytic states of the rotary motor are then compared with the V1 pictures obtained by microsecond scale molecular dynamics simulations. Such comparison allowed to investigate the conformational transitions between catalytic intermediates and provided a unified model of the rotational mechanism. This shows similarities but also differences with that of the F-ATPases, such as the lack of sub-steps during each 120 rotation of the central shaft. Particularly relevant is the finding that, consistently with single-molecule experiments, the simulations revealed that the central shaft undergoes deformations, suggesting that it can store elastic energy. The existence and the need for elasticity in rotary ATPases is further discussed by Colina-Tenorio et al., who present a detailed analysis of the latest high-resolution structures of the peripheral stalk of V-and F-ATPases, and also include A-ATPases, which in archaea function as the F-ATP synthases. These structures have defined the interactions of peripheral stalk subunits and their different conformations, confirming that the peripheral stalk is not static, as initially thought, but rather contributes (in part at least), to the overall enzyme rotational flexibility. An added bonus of this review is the thorough discussion of the role of the peripheral stalk in the process of dimerization of mitochondrial F-ATP synthase. The relevance of conformational changes affecting the peripheral stalk in F-ATP synthase is also covered in the review of Starke et al. These authors discuss how catalysis can be inhibited by binding of the immunomodulatory drug Benzodiazepine (Bz)-423 to the OSCP subunit, which is located on the top of the peripheral stalk. Bz-423 specifically induced apoptosis of autoreactive B-lymphocytes in a murine model of lupus erythematosus, suggesting a promising approach for clinical treatment. The authors carefully describe their progress in defining binding of Bz-423 to OSCP through a wide variety of methods, including screening of a phage display library, genetic removal of OSCP subunit and NMR spectroscopy of purified OSCP and Förster resonance energy transfer in living cells, which allowed to show colocalization of the enzyme with fluorescently tagged Bz-423 with nanometer resolution. The fundamental physiological function of F-ATP synthase in mammals is beautifully described by García-Aguilar and Cuezva. These authors summarize a large body of evidence demonstrating that the F-ATP synthase inhibitor IF1 is a main controller of oxidative phosphorylation under physiological conditions of oxygenation. Based on the findings described in this review, IF1 does only act as an F-ATP synthase inhibitor under conditions of oxygen deficiency and low pH, a view that is still widely held. Transgenic mouse models overexpressing IF1 in colon, liver or neurons displayed IF1-dependent F-ATP synthase inhibition capable of reprogramming energy metabolism to enhanced glycolysis and protection from the action of cytotoxic agents. F-ATP synthase is vital to human health, and its malfunction has been associated to a variety of pathological conditions. These include mutations of mtDNA, which in humans encodes for Fo subunits a and A6L, which contribute to proton transport across the inner membrane. In their fascinating review, Dautant et al. summarize what is currently known about clinical syndromes induced by 58 mutations found in these mitochondriallyencoded genes. Most importantly, they also define the location of these mutations in the most recent high-resolution structures of F-ATP synthase, providing an eye opener on a new level of understanding of how individual mutations may affect enzyme assembly, structure, and catalytic mechanism. V-ATPases are essential for numerous important processes in the eukaryotic cells where they mediate acidification of intraand extra-cellular compartments. Their malfunction is related to several diseases, including cancer. In their very appealing review Collins and Forgac describe the peculiar modes of regulation of enzyme activity by in vivo assembly, i.e., by reversible dissociation of V1 and Vo domains in response to various nutrient cues, such as amino acid or glucose levels; and by enzyme targeting to different cellular membranes, which is controlled by isoforms of Vo subunit a. Most interestingly, these authors demonstrated that overexpression of the a3 isoform is responsible for plasma membrane targeting of V-ATPases in breast tumor cells, which leads to increased propensity to invade. The connections between V-ATPase and glycolysis in cancer is also covered in detail by Hayek et al., who focus in particular on how glucose signaling through V-ATPase acts as a molecular switch that dictates anabolic versus catabolic metabolism in mammalian systems. These glucose-sensing pathways appear to converge at the lysosomes, where a super-complex including aldolase, V-ATPase and the protein kinases AMPK and mTORC1 forms in response to the fructose 1,6 bisphosphate levels. This leads to inactivation of AMPK and activation of mTORC1, promoting cellular anabolism -a finding that can provide new targets against cancer. We are very pleased with these reviews. We hope that our readers will enjoy them as much as we did, and that they will share our enthusiasm for one of the oldest and most important enzymes of life. Frontiers in Physiology | www.frontiersin.org
April 2019 | Volume 10 | Article 358
|
10.1155/2018/8438921
|
cc-by
| null | null |
openalex
|
The objective of this research was to investigate the impact of roadway geometrics and speed on lateral stability of tractor-semitrailer on combined alignments of freeways since the current design guidelines of combined alignments are not available in China. A closed-loop vehicle dynamic simulation model was established using TruckSim multibody dynamics software. The maximum wheel side friction demand and lateral load transfer ratio were used to measure the skidding and rollover risks, and the variation laws of each indicator were determined by developing the controlled variable simulation scenarios. Based on the theory of statistical analysis, an orthogonal test was designed, and the effects of various impact factors on the lateral stability of the tractor-semitrailer were analyzed. The models for lateral stability indicators were developed by multiple linear regression analysis and applied to evaluate the driving risk of tractor-semitrailer on a wet road surface. The results showed that the radius and speed have significant effects on the lateral stability while the effect of the downgrade is of general significance. In addition, lower safe speed should be adopted on wet road surfaces of curved downgrade. This paper proposed a surrogate approach to road safety analysis and the models can be used for building the freeway driving security system.Introduction At present, tractor-semitrailer safety on combined alignments of freeways has been a serious concern in China due to the overloading and relatively poor vehicle performances. According to the survey of serious and major traffic accidents of national highways (2010 to 2014), on freeways of China, the tractor-semitrailer accounted for 38% of all accident vehicles, killing 1,520 people with a mortality rate of 32%. Rearend collision, fixed-object accident, and rollover accident are the main accident types of tractor-semitrailer with the percentage of 73.17% . In the current Chinese highway design standard, the tractor-semitrailer is the newly added design vehicle. No detailed specifications on the configurations of tractor-semitrailer were provided, which required being further studied . Currently, the maximum allowable weight for six-axle tractor-semitrailer is 49000 kg, much higher than the medium truck used in existing highway design and other types of tractor-semitrailer . Meanwhile, the Chinese highway design standard did not specify the design criteria for the combined alignments of freeways [2, 4] . Compared with other truck types, the complex articulated structure, high center of gravity, and the heavy load of six-axle tractor-semitrailer can lead to more severe forms of losing lateral stability when cornering on combined alignments. Therefore, a study is needed to analyze the variation laws of lateral stability of six-axle tractor-semitrailer on combined alignments of freeways. In previous road safety studies, the impact of road alignment parameters on vehicle safety is mainly based on the point-mass model and vehicle dynamics simulation. The side friction factor is commonly used as the indicator of skidding, and the lateral acceleration and lateral load transfer ratio are adopted to measure the risk of rollover. In AASHTO green book, the point-mass model mainly considers the effects of radius and speed on the side friction factor of vehicle on horizontal curves . Since the effect of the longitudinal grade is negligible, Dunlap et al. (1978) developed the equation of side friction factor by analyzing the forces acting on a vehicle on combined grade and horizontal curvature . Eck and French (2002) introduced the wheelbase and wheel angles of tractor and semitrailer to the equation of side friction factor to estimate the required additional superelevation for the tractor-semitrailer at the low radius . However, the principle of the point-mass model is based on the steadystate cornering performance of the rigid vehicle, ignoring the vehicle configuration and characteristics, such as the suspension system, which have a great effect on vehicle rollover as well as the distribution of forces acting on different tires, since friction can vary significantly between tires when cornering . The vehicle dynamics model has the advantages of reflecting the real-time response of the vehicle to evaluate the vehicle operational characteristics under the specific road environments, and the multiple regression analysis was adopted to develop the mathematical models of lateral stability indicators. Kontaratos et al. (1994) developed an enriched bicycle model to study the effects of grade and speed on lateral stability of passenger cars. Since the tractive forces expend some of the lateral friction, the results revealed that the upgrade can have a significant effect on side friction supply and the minimum radius should be increased on upgrades at higher vehicle speeds . Bonneson (2000) found that the downgrade depletes the margin of safety for heavy trucks traveling on downgrade horizontal curves owing to the great weight and high peak side friction demands of heavy trucks . You and Sun (2013) developed the driver-vehicleroad closed-loop dynamic simulation model of a two-axle convention truck and found that the rollover is the main concern for trucks on horizontal curves, which is affected by the vertical curve, small radius, and steep slope . Kordani et al. (2014 Kordani et al. ( , 2015) ) used CarSim and TruckSim to examine the effect of combined alignments on lateral stability of passenger cars and two-axle conventional trucks and presented a series of formulas based on the multiple regression analysis. They found that a higher maximum wheel side friction demand is produced on steep downgrades and the truck is more likely to roll over [14, 15] . Shin and Lee (2015) investigated the lateral stability of heavy trucks by TruckSim and the study suggested that the rollover is highly sensitive to vehicle speed and steering angle, whereas skidding is vulnerable to the change in tire-road friction coefficient . Zhang et al. (2015) studied the lateral stability of passenger cars on horizontal curves by CarSim and developed the mathematical models of lateral stability with the consideration of curvature radius, longitudinal grade, speed, and brake pressure . Wang et al. (2015) adopted the multiple linear regression models to analyze the effects of the combined alignments on the lateral acceleration of passenger cars . By focusing on the dynamic performances of trucks, a series of researches were conducted to put forward the recommendations to the existing roadway design policies. Considering the driver comfort levels and necessity to maintain the vehicle safety, the effect of vertical alignment on minimum radius requirements was evaluated using computer simulation and multiple linear regression method was employed to calibrate the minimum radius requirements for combined alignments based on the AASHTO green book . Torbic et al. ( 2014 ) used a combination of field data and analytical and vehicle dynamics simulation models to evaluate geometric design criteria for sharp horizontal curves on steep grades and put forward the limit of maximum superelevation rate on a simple horizontal curve combined with vertical alignments . Li and He (2016) demonstrated the safety margins of lateral tire forces for radius, operating speed, and superelevation rate on horizontal curves to guarantee good vehicle lateral reliability and ride comfort . The previous studies mainly concentrated on lateral stability of passenger cars and two-axle conventional trucks according to the AASHTO roadway design policy but paid little attention to the lateral stability of six-axle tractorsemitrailer on combined alignments of freeways. To analyze and reveal the traffic accident mechanism of rollover and skidding of six-axle tractor-semitrailer, the present study is organized as follows. In Section 2, a driver-vehicle-road closed-loop model considering three-dimensional alignment was established by TruckSim. In Section 3, to demonstrate the effects of speed and roadway geometrics on lateral stability of the vehicle, the controlled variable method and orthogonal test method were used to develop the simulation scenarios, and the one-way analysis of variance was adopted to analyze the effects of various factors quantitatively. In Section 4, the simulation results were used to establish the models for lateral stability indicators and put forward a speed limit for six-axle tractor-semitrailer on the wet road surface.
Materials and Methods TruckSim multibody vehicle dynamics software was used to establish the driver-vehicle-road dynamic simulation model. It consists of three essential components: the driver control model, the vehicle model, and the 3D road model. A typical six-axle tractor-semitrailer was used to evaluate the stability performance on different terrains at various speeds. The detailed parameter settings and establishment process of the model were described in this section. Based on force analysis of tractor-semitrailer on combined alignments, the evaluation indicators of lateral stability were determined to measure the risk of skidding and rollover.
Driver-Vehicle-Road Dynamic Simulation Model 2.1.1. Vehicle Model. The investigated tractor-semitrailers selected are Suzuki QL4250SKFZ three-axle tractor and Huajun ZCZ9390 three-axle semitrailer, which are commonly used in China. The drive type of the tractor is 6 × 4. The gross vehicle weight is 49000 kg with a rated load of 33000 kg. The vehicle model includes four-wheel drive powertrain, air pressure braking system, Ackermann steering mechanism, solid axle suspensions of steering axle, drive axles, and trailer axles. According to the structural parameters of the investigated tractor-semitrailer and the field test to calibrate the vehicle model, the main parameters of tractor-semitrailer were determined, as shown in Table 1 . The vehicle configuration interfaces in TruckSim are shown in Figure 1 . friction of the ground relative to a 3D reference line. The geometric alignments consist of the horizontal alignment, vertical alignment, and cross section. In TruckSim, the "X-Y coordinates of centerline" module defines the horizontal alignment of the road, using a table of X-Y coordinates of the investigated road segment calculated by multiquadric (MQ) interpolation method. For the vertical alignment design, the "centerline elevation: Z versus S" module is available for specifying the elevation for the reference line through inputting the elevation of the starting and ending points of the road segment. The superelevation rate of the cross section is set by modifying the elevations of both sides of the road segment at the feature points such as the straightspiral point, spiral-curve point, curve-spiral point, and spiraltangent point. For the dry asphalt pavement, the typical value of friction coefficient is 0.8 (Yu, 2011) . Figure 2 shows an example of the detailed settings of 3D road model in TruckSim simulation.
Driver Control Model. The closed-loop driver control model contains four parts: speed control, brake control, gearshifting control, and steering control. The vehicle travels at a constant target speed. Automatic gear-shifting control was adopted using the closed-loop shift control at all gears. For the downgrades, the step brake control was used to maintain the constant descending speed, and the values of braking force varied along with the simulation step size. In steering control, the key control parameters are the target path of the vehicle and the preview time. The vehicle travels along the road centerline as the target path with no offset. The preview control looks ahead a distance determined from the specified time and the current speed, to compare the current vehicle trajectory to the target path. A realistic value for the preview time is about 1.5 seconds, which can produce smoother steering to keep the vehicle on the target winding paths.
Experimental Validation. To further investigate the accuracy of the driver-vehicle-road dynamic simulation model, the field test was conducted on the Hunchun-Dunhua freeway, which is a segment of G12 National Highway, in Jilin province, China. The tractor-semitrailer was controlled by a driver with more than 10 years of experience. The speed and lateral acceleration were recorded by VBOX and gyroscope simultaneously, and the experimental setup and test instruments are shown in Figure 3 . One typical segment with curve radius of 490 m and average gradient of 3.1% (-3.1% for downgrade) was selected, and the lengths of the spiral transition curve and circular curve section are 120 m and 305 m, respectively. In the process of field test, owing to the effect of real-time traffic status, the initial speeds for upgrade and downgrade were 86.3 km/h and 67.9 km/h, respectively. The comparisons of simulated and measured results on curved upgrade and downgrade are shown in Figure 4 . As shown in Figure 4 (a), on the curved upgrade, with the increase of the length of longitudinal slope, the simulated speed and measured speed decrease gradually. The simulated and measured lateral acceleration reaches 0.11 g at the spiral-curve point. On the circular curve section, the lateral acceleration decreases slowly; after entering the spiral transition curve, the lateral acceleration decreases rapidly with the decrease of curvature. As shown in Figure 4 (b), on the curved downgrade, the simulated and measured speeds increase slightly with the increase of longitudinal grade. On the circular curve section, the lateral acceleration obtained from the simulation and field test increases slowly, reaching the maximum value of 0.08 g at the curve-spiral point. After entering the spiral transition curve, the values of lateral acceleration decrease rapidly. It can be concluded that the dynamic responses of the simulation model were in good agreement with those of field tests, indicating that the driver-vehicleroad dynamic simulation model was sufficiently accurate to analyze the lateral stability of the tractor-semitrailer.
Force Analysis of Combined Alignments. A three-dimensional reference system of the road was developed to analyze the force condition under the influence of cross slope and the longitudinal slope. The original reference system (X 0 , Y 0 , Z 0 ) is defined as the reference system of the horizontal curve without superelevation, where O is the coordinate origin fixed on the ground. Then, by rotating coordinators with the degree of θ around the Y 0 axis, the reference system on vertical alignment (X θ , Y θ , Z θ ) was developed, and θ is the angle of vertical slope. Finally, through rotating the coordinators with the degree of α (the angle of cross slope) around the X 0 axis, the three-dimensional reference system for combined horizontal and vertical alignments (X r , Y r , Z r ) was developed, as shown in Figure 5 . The reference transformation formula from the original reference system (X 0 , Y 0 , Z 0 ) to the threedimensional reference system (X r , Y r , Z r ) can be expressed as [ [ [ X r Y r Z r ] ] ] = [ [ [ cos θ 0 sin θ sin α sin θ cos α -sin α cos θ -sin θ cos α sin α cos α cos θ ] ] ] [ [ [ X 0 Y 0 Z 0 ] ] ] . (1) Figure 5 shows the three-dimensional reference system and the force analysis of tractor-semitrailer cornering on combined horizontal and vertical alignments. In Figure 5 , F is the centrifugal force (N), G is the vehicle gravity (N), F y is the total lateral tire force (N), F z is the total vertical tire force (N), F yLno , F yLni , F yRno , and F yRni are lateral forces of the outer and inner tires of the left and right nth axle (N), F zLno , F zLni , F zRno , and F zRni are vertical forces of the right nth axle (N), g is the gravity acceleration (m/s 2 ), V is the speed (km/h), R is the radius of curve (m), α is the angle of cross slope ( ∘ ), and θ is the angle of vertical slope ( ∘ ). A general formula of lateral tire forces and vertical tire forces acting on six-axle tractor-semitrailer can be written as F = GV 2 gR , F y = 6 ∑ n=1 (F yLno + F yLni + F yRno + F yRni ) = GV 2 gR cos α -G cos θ sin α,
Vertical direction Perpendicular to the roadway Tangential to the roadway, perpendicular to the driving direction Tangential to the roadway, along the driving direction Z 0 Z Z r O X 0 X (X r ) Y r Y 0 (Y ) (a) Three-dimensional reference system Z 0 Z r e F F y F z G Roll center h g h 0 Y r b F yLno F zLno F yLni F zLni F yRni F yRno F z = 6 ∑ n=1 (F zLno + F zLni + F zRno + F zRni ) = GV 2 gR sin α + G cos θ cos α. (2)
Evaluation Indicator of Lateral Stability. The skidding and rollover are the main hazardous situations of the sixaxle tractor-semitrailer losing lateral stability on the curved upgrades and downgrades. The vehicle skids when the centrifugal force on the mass center of the vehicle exceeds the lateral forces between the tire and the pavement and rolls over when the centrifugal force moment exceeds the gravity moment. The high speed, heavy load, and decreased road friction coefficient are the primary cause of the skidding and rollover. According to Figure 5 , the maximum wheel side friction demand was selected as the evaluation indicator of skidding. The definition of maximum wheel side friction demand is the absolute value of the maximum ratio of total lateral forces to total vertical forces of each side of axle, denoted by f D . It is also the maximum wheel side friction demand by the vehicle moving on the curved road without skidding, which can be given by [ where f D denotes the maximum wheel side friction demand, F yLno and F yLni are lateral forces of the outer and inner tires of the left nth axle (N), F yRno and F yRni are lateral forces of the outer and inner tires of the right nth axle (N), F zLno and F zLni are vertical forces of the left nth axle (N), and F zRno and F zRni are the vertical forces of the right nth axle (N). The vehicle will skid if the maximum wheel side friction demand f D exceeds the supplied road friction coefficient φ, so the value of road friction coefficient φ is the safety threshold of maximum wheel side friction demand f D . The vehicle rollover is one of the most frequent single vehicle accidents, especially for six-axle tractor-semitrailers with a high gravity center. The truck experiences vertical load transfer from the tires on one side of the vehicle to those on the other side when traveling on curved road sections. The lateral load transfer ratio denoted by LTR is used to measure the risk of the rollover of the heavy truck [25, 26] . It is defined as the ratio of the difference between the sum of right wheel loads and the sum of left wheel loads to the sum of total wheel loads , as shown in LTR = ∑ 6 n=1 (F zLno + F zLni ) -∑ 6 n=1 (F zRno + F zRni ) ∑ 6 n=1 (F zLno + F zLni ) + ∑ 6 n=1 (F zRno + F zRni ) , (4) where LTR denotes the lateral load transfer ratio, F zLno and F zLni are vertical forces of the left nth axle (N), and F zRno and F zRni are the vertical forces of the right nth axle (N). Based on the research conducted by Fan et al. ( 2016 ), the risk level of LTR can be classified into three states : (1) When LTR ∈ [0.6, 0.8), the vehicle has a modest potentiality in occurrence of rollover, which is defined as the warning state of rollover. (2) When LTR ∈ [0.8, 1), the vehicle has a definite potentiality in occurrence of rollover; this state is defined as dangerous state of rollover. (3) When LTR = 1, the wheels of one side leave the ground, and the vertical load of one side transfers to the wheels on the other side; this state is defined as "critical state of rollover." Tangent-spiral point
Midpoint of curve Curve-spiral point U pg ra de + 3% , + 4% , + 5% , + 6% D ow ng ra de -3 % , -4 % , -5 % , -6 % Spiral-curve point Spiral-tangent point
Simulation Process and Driving Dynamics Analysis This section describes the criteria used for the selection of road alignments; a series of controlled variable simulation scenarios and orthogonal tests were designed. The variation laws of lateral stability indicators in controlled variable simulation scenarios and the ANOVA results of orthogonal tests regarding the roadway geometrics and speeds were presented.
Design of Controlled Variable Simulation Scenarios. To explore the variation laws of f D and LTR along the stations under the influence of roadway geometrics and speeds, respectively, a series of controlled variable simulation scenarios were developed. For the determination of the road alignments, according to the Chinese standard Technical Standard of Highway Engineering (JTGB01-2014) and Design Specification for Highway Alignment (JTGD20-2006) , the freeway with a design speed of 80 km/h was considered. For the horizontal alignment, the ultimate minimum radius is 250 m with a maximum superelevation rate of 8%. To compare the impact of the radius on the lateral stability, a control group with the radii of 450 m, 650 m, and 850 m were selected. The total length of the simulated segment is 800 m: the length of the approach and departure tangent is 100 m, the length of the spiral transition curve L s is 200 m, and the length of the circular curve is 200 m. For the vertical alignment, the maximum allowable longitudinal grade of freeway with the design speed of 80 km/h is 6%, and therefore the upgrades of 3%, 4%, 5%, and 6% and downgrades of -3%, -4%, -5%, and -6% were selected. In the standard, the critical slope length increases with the decreasing longitudinal grade. To simplify the influence of the slope length, the longitudinal slope starts at the distance of 150 m and ends at the distance of 650 m with a total length of 500 m. The road alignment parameters of scenarios are shown in Figure 6 , and the detailed design of controlled variable simulation scenarios is presented in Table 2 .
Simulation Results of Controlled Variable Simulation Scenarios. Figure 7 shows the simulation results of the variation laws of f D and LTR on curved upgrades. It can be found that f D and LTR present the same variation law. On the approach tangent (from 0 to 100 m), the values of f D and LTR are approximate to 0. As the vehicle enters the spiral transition curve (from 100 m to 300 m), the centrifugal force increases as the curvature radius reduces gradually, and the values of f D and LTR increase dramatically. As the length of longitudinal slope increases, the speed decreases on upgrades, and the values of f D and LTR decrease slightly. On the circular curve (from 300 m to 500 m), owing to the smallest curvature radius, the constant maximum centrifugal force causes the maximum roll motion of the vehicle, and therefore f D and LTR reach the peak values. However, the constant horizontal radius makes constant and limited contribution to the roll motion of the vehicle; due to the speed loss of the upgrades, the values of f D and LTR decrease sharply. On the spiral transition curve (from 500 m to 700 m), as the curvature radius increases gradually and the ascending speed decreases, the centrifugal force on the truck slowly decreases. Therefore, the values of f D and LTR show a dramatic reduction. After entering the departure tangent (from 700 to 800 m), the values of f D and LTR are approximate to 0, without the influence of horizontal curve and upgrades. The variation laws reveal that the values of f D and LTR are in direct proportion to the speed while being in inverse proportion to the radius and upgrade. Figure 8 shows the simulation results of the variation laws of f D and LTR on curved downgrades. The values of f D and LTR on curved downgrades have a similar tendency to the curved upgrades except in the circular curve section ) orthogonal array was selected to analyze the three factors with four levels for curved upgrades and downgrades, respectively. Factors and corresponding levels are shown in Table 3 . In the column of grade, the negative gradients in brackets denote the downgrades. F-test was used for significance testing of factors in the variance analysis. The significance level α includes 0.01, 0.05, and 0.10 for F distribution. For the factor i, if F i >= F 0.01 , the factor i has a high significant effect on the dependent variables; if F 0.05 <= F i < F 0.01 , the factor i has a significant effect on the dependent variables; if F 0.1 <= F i < F 0.05 , the factor i has a general significant effect on the dependent variables; if F i < F 0.1 , the factor i has no significant effect on the dependent variables. Tables 4 and 5 show the variance analysis of factors influencing f D and LTR on curved upgrades. It can be found that, on curved upgrades, the order of primary and secondary factors affecting f D and LTR is radius, speed, and grade according to the order of sum of squares. The F values of radius and speed are greater than the corresponding critical F values, and therefore the radius has a highly significant effect on the lateral stability of tractor-semitrailer, and speed has a significant effect whereas the effect of the grade is not significant. Tables 6 and 7 show the variance analysis of factors influencing f D and LTR on curved downgrades. It can be found that, on curved downgrades, the values of sum of squares decrease gradually from the radius to speed to grade, indicating that the order of primary and secondary factors affecting f D and LTR is the radius, speed, and grade, respectively. The F values of the radius, speed, and grade are greater than the corresponding critical F values, and therefore the effect of the radius is of high significance, the effect of the speed is of significance, and the effect of the grade is of general significance. This is mainly due to the fact that the radius and speed can influence the roll motion of vehicle and centrifugal force directly when cornering on curves. The grade can change the component of force, as its value is relatively low, so the effect of the grade is less significant than radius and speed.
Results and Discussion Following the consideration of the simulation results, the multiple linear regression analysis was conducted to develop new models of f D and LTR for six-axle tractor-semitrailer by selecting the radius, longitudinal grade, and vehicle speed as the independent variables. Based on the models, the risk analysis of vehicle on the wet road surface was conducted and the safe speed limits were put forward.
Calibrated Mathematical Model. A total of 56 observations of curved upgrades and downgrades were used to calibrate the mathematical model. Many independent variables were examined, and the final models were calibrated based on the following criteria : (1) The coefficient of determination R 2 must be significant at the 0.95 confidence level. (2) Each of the independent variables used in the model must have a coefficient that is significantly different from zero at the 0.95 confidence level. (3) The algebraic signs of the coefficients of the independent variables must have a logical explanation. The final models of f D and LTR on curved upgrades and downgrades were presented in ( 5 )-( 6 ). where LTR is the lateral load transfer ratio; R is the radius (m); i is the grade (%); V is the speed (km/h). From Tables 8 and 9 , the P value of all independent variables is significantly different from zero at the 95% confidence level, so ( 5 )-( 6 ) have logical explanations for the effect of each independent variable on dependent variables, f D and LTR. The positive sign for the coefficient of V 2 /R means that f D and LTR increase with the increase in V 2 /R. The negative sign for the coefficient of i means that, on curved upgrades, f D and LTR decrease as grade i increases. On curved downgrades, f D and LTR increase when downgrade i increases (as the signs for downgrades are negative).
Risk Analysis of Vehicle on Wet Road Surface. On wet road surface, the road friction coefficient is greatly decreased by rainwater. Meanwhile, the rainwater that accumulated in the tread pattern may lead to a decrease in the contact area between the tire and the pavement. As a result, brake failure and inflexible steering can greatly reduce the lateral stability of the vehicle, and the high speed can lead to the increase in centrifugal force, increasing the probability of traffic accidents. Therefore, by conducting the risk analysis of the vehicle on a wet road surface, the safe speed limit is put forward. The supplied friction coefficient φ of wet road surface at different speeds V can be determined as follows (Wambold et al., 1984) : φ = 0.874e -0.00393V . (7) The lateral stability of the vehicle on curved upgrades and downgrades can be analyzed by utilizing ( 5 )- (6) . By comparing the values of f D and LTR with the available friction coefficient φ and the rollover risk level, respectively, the risk analysis on wet road surface was conducted. For the freeway with the design speed of 80 km/h, the ultimate minimum horizontal curve radius of 250 m and general minimum horizontal curve radius of 400 m with the superelevation rate of 8% were selected as the evaluating section. The upgrade ranges from 3% to 6%, and the downgrade ranges from -3% to -6%. The six-axle tractor-semitrailer travels at the speed of 70 km/h to 90 km/h. The risk analyses of vehicle on upgrades and downgrades combined with the ultimate minimum horizontal curve radius and the general minimum horizontal curve radius were presented in Tables 10 and 11 , respectively. From Tables 10 and 11 , the following can be concluded. (1) To avoid the skidding risk, the safe speed on the curved upgrade is higher than that on curved downgrades. For the radius of 250 m combined with upgrades of 3%, 4%, and 5%, the skidding occurs at 90 km/h. There is no skidding risk of tractorsemitrailer at 90 km/h on upgrade of 6%. On curved downgrades, the skidding occurs at 90 km/h on downgrades of -3% and -4% and 85 km/h on downgrades of -5% and -6%. (2) From the viewpoint of the rollover risk, the risk levels on the downgrades are higher than on upgrades. On curved upgrades, for the ultimate minimum radius of 250 m combined with upgrades of 3%, 4%, and 5%, at the speed of 70 km/h, the vehicle is in the safe status. However, when the speed reaches 75 km/h or more, the vehicle remains in the warning state of rollover. On the upgrade of 6%, the vehicle is in the warning state of rollover within a range from 80 km/h to 90 km/h. On curved downgrades of -3% and -4%, the vehicle is in the warning state of rollover within a (3) The lateral stability of tractor-semitrailer improves markedly on the general minimum radius of 400 m. On upgrades, the values of f D and LTR are much lower than the threshold values, so the six-axle tractor-semitrailer remains in safe status at the speed of 90 km/h. On downgrades of -3%, -4%, -5%, and -6%, the speed causing the warning state of rollover is 90 km/h, 85 km/h, 85 km/h, and 80 km/h, respectively. Therefore, the general minimum radius can basically satisfy the lateral stability requirements of six-axle tractor-semitrailer at the design speed of 80 km/h. Besides, it is necessary to limit the speed on the ultimate minimum horizontal curve radius combined with upgrades and downgrades.
Conclusions To investigate the lateral stability of six-axle tractor-semitrailer on combined alignments of the freeway, the closed-loop vehicle dynamic simulation model using TruckSim multibody software was established, and the following remarks are offered: (1) The variation laws of the maximum wheel side friction demand f D and lateral load transfer ratio LTR on curved upgrades are consistent with the curved downgrades except in circular curve section due to the speed loss on curved upgrades. The steeper upgrade causes a reduction in f D and LTR whereas the steeper downgrade can lead to an increase in f D and LTR, showing that more side friction demand and roll motion are created on steep downgrades compared with upgrades and mild downgrades. (2) On both curved upgrades and downgrades, the order of primary and secondary factors affecting f D and LTR is radius, speed, and grade. The effect of the horizontal radius is of high significance, and the effect of speed is of significance. On curved downgrades, the grade has the general significant effect on lateral stability of six-axle tractor-semitrailer. Therefore, more concerns should be attached to the safety issues of curved downgrades. (3) The horizontal radius, speed, and grade were introduced to calibrate the mathematical models of the lateral stability. Skidding and rollover risks were analyzed on the wet road surface of the curved upgrades and downgrades, respectively. This study recommends that, to ensure the lateral stability of six-axle tractor-semitrailer on the wet road surface, low speed should be adopted to ensure the lateral stability of tractor-semitrailer. From the comprehensive perspective of preventing the skidding risk and rollover risk, for the ultimate minimum radius, the safe ascending speed and descending speed should better not exceed 75 km/h and 70 km/h, respectively; for the ultimate minimum radius of 400 m, a safe descending speed should better not exceed 80 km/h. (4) The results and recommendation models can provide guide for both combined alignments design and safety improvement of existing roads. However, this research only considered the lateral stability of six-axle tractor-semitrailer on the simple horizontal curves combined with vertical slopes. Therefore, the lateral stability related to more alignment combinations such as the compound horizontal curves and reverse horizontal curves on upgrades and downgrades should be investigated in further research. 2. 1 . 2 . 12 3D Road Model. The 3D road model in TruckSim simulation software defines the geometric alignments and
Figure 1 : 1 Figure 1: Vehicle configuration interfaces in TruckSim.
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 0 Global X coordinate (m) (a) Settings of horizontal alignment 200 250 300 350 400 450 500 550 600 650 700 800 0 Station (m) (c) Settings of cross section
Figure 2 : 2 Figure 2: Settings of 3D road model in TruckSim simulation.
Figure 3 :Figure 4 : 34 Figure 3: Experimental setup and test instruments.
FFigure 5 : 5 Figure 5: Force analysis of tractor-semitrailer cornering on combined alignments.
Figure 6 : 6 Figure 6: Road geometric parameters of scenarios.
5 ( 5 Variation laws of f D and LTR of different curvature radii Maximum demand pavement friction coefficient f D Lateral-load transfer ratio (LTR) Variation laws of f D and LTR of different grades Maximum demand pavement friction coefficient f D V = 60 km/h V = 70 km/h V = 80 km/h V = 90 km/h Lateral-load transfer ratio (LTR) V = 60 km/h V = 70 km/h V = 80 km/h V = 90 km/c) Variation laws of f D and LTR of different speeds
Figure 7 : 6 ( 76 Figure 7: Variation laws of f D and LTR on curved upgrades.
Figure 8 : 8 Figure 8: Variation laws of f D and LTR on curved downgrades.
Table 1 : 1 Main parameters of tractor and semitrailer. Category
Table 2 : 2 Design of controlled variable simulation scenarios. Road type Scenarios Radius (m) Grade (%) Speed (km/h) Simulation purpose 1 250, 450, 650, 850 4 80 Study the effect of radius Curved upgrades 2 450 3, 4, 5, 6 80 Study the effect of grades 3 450 4 60, 70, 80, 90 Study the effect of speeds 1 250, 450, 650, 850 -4 8 0 S t u d y t h e e ff e c t o f r a d i u s Curved downgrades 2 4 5 0 -3, -4, -5, -6 80 Study the effect of grades 3 4 5 0 -4 60, 70, 80, 90 Study the effect of speeds
Table 3 : 3 Factors and corresponding levels. Level Radius (m) Grade (%) Speed (km/h) 1 2 5 0 3 ( -3) 60 2 4 5 0 4 ( -4) 70 3 6 5 0 5 ( -5) 80 4 8 5 0 6 ( -6) 90 (from 250 m to 450 m). Instead of the decreasing values of f D and LTR on upgrades, f D and LTR reach the peak values after entering the circular curve, as the braking forces act on the vehicle to maintain a constant speed, and there is no speed loss on downgrades; the values of f D and LTR remain constant. Particularly, on the circular curve, there is an increase in values of f D and LTR on downgrades of -5% and -6%, indicating that, on downgrades steeper than -4%, the descending speed still increases slightly though the braking maneuvers are applied. The variation laws reveal that, in braking cases, the values of f D and LTR are in direct proportion to speed and downgrade while being in inverse proportion to the radius. 3.3. Design and ANOVA Results of Orthogonal Tests. The orthogonal test method is commonly used in experimental study of multilevel factors by effectively simplifying the design of experiment test. In this research, it can be used to quantitatively analyze the effects of multilevel factors on the lateral stability of six-axle tractor-semitrailer on curved upgrades and downgrades. The L 16 (4 5
Table 4 : 4 Variance analysis of factors influencing f D on curved upgrades. Source Sum of squares df Mean square F value Critical F value Corrected model 0.186 9 0.021 32.686 Intercept 1.731 1 1.731 2731.354 Radius 0.146 3 0.049 77.008 F 0.01 (3, 3) = 29.5 Grade 0.001 3 0.000 0.414 F 0.1 (3, 3) = 5.39 Speed 0.039 3 0.013 20.637 F 0.05 (3, 3) = 9.28 Error 0.004 6 0.001 Total 1.921 16 Correct total 0.190 15
Table 5 : 5 Variance analysis of factors influencing the LTR on curved upgrades. Source Sum of squares df Mean square F value Critical F value Corrected model 0.243 9 0.027 34.915 Intercept 3.042 1 3.042 3926.216 Radius 0.178 3 0.059 76.416 F 0.01 (3, 3) = 29.5 Grade 0.001 3 0.000 0.507 F 0.1 (3, 3) = 5.39 Speed 0.065 3 0.022 27.821 F 0.05 (3, 3) = 9.28 Error 0.005 6 0.001 Total 3.291 16 Correct total 0.248 15
Table 6 : 6 Variance analysis of factors influencing f D on curved downgrades. Source Sum of squares Df Mean square F value Critical F value Corrected model 0.325 9 0.036 21.211 Intercept 1.951 1 1.951 1146.316 Radius 0.208 3 0.069 40.808 F 0.01 (3, 3) = 29.5 Grade 0.035 3 0.012 6.873 F 0.1 (3, 3) = 5.39 Speed 0.081 3 0.027 15.953 F 0.05 (3, 3) = 9.28 Error 0.010 6 0.002 Total 2.286 16 Correct total 0.335 15
Table 7 : 7 Variance analysis of factors influencing the LTR on downgrades. Source Sum of squares df Mean square F value Critical F value Corrected model 0.480 9 0.053 23.119 Intercept 4.197 1 4.197 1819.425 Radius 0.301 3 0.100 43.538 F 0.01 (3, 3) = 29.5 Grade 0.052 3 0.017 7.524 F 0.1 (3, 3) = 5.39 Speed 0.127 3 0.042 18.294 F 0.05 (3, 3) = 9.28 Error 0.014 6 0.002 Total 4.691 16 Correct total 0.494 15 Curved upgrades: f D = 0.015 Curved downgrades: f D = 0.018 V 2 R V 2 R -0.013i + 0.194 -0.02i + 0.022, (5) Curved upgrades: LTR = 0.017 Curved downgrades: LTR = 0.022 V 2 R -0.025i + 0.112, V 2 R -0.014i + 0.281 where f D is the maximum wheel side friction demand; R is the radius (m); i is the grade (%); V is the speed (km/h).
Table 8 : 8 Results of statistical analysis for f D cornering on upgrades and downgrades. Road type Independent variable B t P value Constant 0.194 26.441 0.001 Curved upgrades V 2 /R i 0.015 -0.013 63.064 -7.976 0.001 0.001 R 2 = 0.994 Constant 0.022 2.635 0.015 Curved downgrades V 2 /R i 0.018 -0.020 68.886 -11.326 0.001 0.001 R 2 = 0.996
Table 9 : 9 Results of statistical analysis for LTR cornering on upgrades and downgrades. Road type Independent variable B t P value Constant 0.281 60.183 0.001 Curved upgrades V 2 /R i 0.017 -0.014 113.230 -14.143 0.001 0.001 R 2 = 0.998 Constant 0.112 8.347 0.001 Curved downgrades V 2 /R i 0.022 -0.025 50.799 -8.792 0.001 0.001 R 2 = 0.992
Table 10 : 10 Risk Analysis of Tractor-semitrailer on the Ultimate Minimum Horizontal Curve Radius Combined with Upgrades and Downgrades. Risk status Warning state of rollover Warning state of rollover Warning state of rollover Dangerous state of rollover Skidding, dangerous state of rollover Warning state of rollover Warning state of rollover Warning state of rollover Dangerous state of rollover Skidding, dangerous state of rollover Warning state of rollover Warning state of rollover Dangerous state of rollover Skidding, dangerous state of rollover Skidding, dangerous state of rollover Warning state of rollover Warning state of rollover Dangerous state of rollover Skidding, dangerous state of rollover Skidding, dangerous state of rollover LTR 0.62 0.68 0.75 0.82 0.90 0.64 0.71 0.78 0.85 0.92 0.67 0.73 0.80 0.87 0.95 0.67 0.73 0.83 0.90 0.97 f D 0.43 0.49 0.54 0.60 0.67 0.45 0.51 0.56 0.62 0.69 0.47 0.53 0.58 0.64 0.71 0.47 0.53 0.60 0.66 0.73 φ 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 V/(km/h) 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 i/% -3 -4 -5 -6 Risk status Safe Warning state of rollover Warning state of rollover Warning state of rollover Skidding, warning state of rollover Safe Warning state of rollover Warning state of rollover Warning state of rollover Skidding, warning state of rollover Safe Warning state of rollover Warning state of rollover Warning state of rollover Skidding, warning state of rollover Safe Safe Warning state of rollover Warning state of rollover Warning state of rollover LTR 0.57 0.62 0.67 0.73 0.79 0.56 0.61 0.66 0.72 0.78 0.54 0.59 0.65 0.70 0.76 0.53 0.58 0.63 0.69 0.75 f D 0.45 0.49 0.54 0.59 0.64 0.44 0.48 0.53 0.58 0.63 0.42 0.47 0.51 0.56 0.62 0.41 0.45 0.50 0.55 0.60 φ 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 V/(km/h) 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 i/% 3 4 5 6
Table 11 : 11 Risk analysis of tractor-semitrailer on the general minimum horizontal curve radius combined with upgrades and downgrades. from 70 km/h to 80 km/h and dangerous state of rollover within a range from 85 km/h to 90 km/h. On curved downgrades of -5% and -6%, the vehicle remains in the warning state of rollover within a range from 70 km/h to 75 km/h and dangerous state of rollover within a range from 80 km/h to 90 km/h. Risk status Safe Safe Safe Safe Warning state of rollover Safe Safe Safe Warning state of rollover Warning state of rollover Safe Safe Safe Warning state of rollover Warning state of rollover Safe Safe Warning state of rollover Warning state of rollover Warning state of rollover LTR 0.46 0.50 0.54 0.58 0.63 0.48 0.52 0.56 0.61 0.66 0.51 0.55 0.59 0.63 0.68 0.53 0.57 0.61 0.66 0.71 f D 0.30 0.34 0.37 0.41 0.45 0.32 0.36 0.39 0.43 0.47 0.34 0.38 0.41 0.45 0.49 0.36 0.40 0.43 0.47 0.51 φ 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 V (km/h) 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 i/% -3 -4 -5 -6 Risk status Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe Safe LTR 0.45 0.48 0.51 0.55 0.58 0.43 0.46 0.50 0.53 0.57 0.42 0.45 0.48 0.52 0.56 0.41 0.44 0.47 0.50 0.54 f D 0.34 0.37 0.40 0.43 0.46 0.33 0.35 0.38 0.41 0.45 0.31 0.34 0.37 0.40 0.43 0.30 0.33 0.36 0.39 0.42 φ 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 0.66 0.65 0.64 0.63 0.61 V (km/h) 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 70 75 80 85 90 i/% 3 4 5 6 range
|
10.1016/j.ejps.2020.105269
|
cc-by
| null | null |
openalex
|
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Introduction Microneedles are intensively studied for dermal and transdermal drug and vaccine delivery (1, 2) . Microneedles are needle-like structures up to 1 mm in length, capable of piercing the upper layer of the skin, the stratum corneum, enabling drug and vaccine delivery in a minimally invasive and painless way (3, 4) . Several microneedle concept are in development: i) hollow microneedles to inject liquid formulations through the bore of the microneedle, ii) microneedles for skin pretreatment to create microchannels prior application of a patch containing the drug or vaccine formulation, iii) porous, coated, hydrogel-forming microneedles and dissolving microneedles that release the drug or vaccine upon insertion into the skin (5) (6) (7) (8) . Dissolving microneedles (dMNs), completely dissolve after insertion into the skin, thereby releasing the encapsulated drug or vaccine (3) . As an additional advantage, the dry state of dMNs combined with the presence of excipients can increase protein thermostability (3) . dMNs are produced from different materials, ranging from low molecular weight carbohydrates to biodegradable polymers (3) . The selection of the matrix material is based on different factors such as i) safety, ii) compatibility with the active compound and the manufacturing procedure, iii) capability of the manufactured dMNs to efficiently pierce the skin and subsequently dissolve in a short time period and iv) potential adjuvant properties. One of the most frequently used matrix material is the hyaluronan (HA) (3, (9) (10) (11) (12) (13) . High molecular weight HA is a non-toxic, biodegradable, biocompatible and non-inflammatory linear polysaccharide (14) naturally present in the skin and approved by the FDA as inactive material. Although frequently used for microneedle manufacturing, the molecular weight of the polysaccharide HA is rarely addressed (1, 3, 9, 11, 12, (15) (16) (17) . There are indications however, that the molecular weight of the HA influences its immuno-adjuvant properties. Only high molecular weight HA (HMW-HA, MW 10 7 Da) is an ubiquitous extracellular matrix component (18, 19) . Low molecular weight HA (LMW-HA) (4-to 16-oligosaccharide size, 800 -3200 Da (19, 20) ) is able to activate immunocompetent cells such as macrophages (21) and DCs (19) (20) (21) (22) (23) . These activated human DCs are able to stimulate T-cell mediated immune responses (19, 20, 24) . The immune activation is mediated by the HA binding to specific receptors such as CD44, CD168, Toll-like receptor (TLR)-2 and TLR-4 (25) (26) (27) (28) . The latter is a receptor associated with the innate immunity and the host defence against bacterial infection (20) . In the presence of antigens, a specific response may develop (29) and so the LMW-HA can act as an adjuvant during vaccination. In a previous study from our group, the dissolving microneedle properties of the only 150 kDa HA, such as penetration and dissolution in the skin, have been reported (30) . The aim of this study was to determine whether i) HA with different MW had an effect on the immune response for both antibody (in vivo injections by hMN) and cellular (in vitro T-cell exposure) responses and ii) it was possible to fabricate dMNs by micromolding from HA with different molecular weight. To this end, first the potential adjuvant effect of HA was assessed by i) performing immunization studies investigating the antibody response in mice injected with the model antigen ovalbumin (OVA) in presence of a range of different HA-MWs and ii) investigating T-cell activation in vitro upon exposure to several HA-MWs with OVA. Subsequently, different HA-MWs were used to fabricate dMNs assessing the compatibility of the matrix material of each MW with the manufacturing procedure. These dMNs were used to investigate their capability to pierce the skin and to dissolve in reasonable short time period.
Materials and methods
Materials Hyaluronan (HA) (sodium hyaluronate, average Mw was 4.8 kDa, 20 kDa and 150 kDa (endotoxin level <= 0.05 EU/mg) or 1.8 MDa (endotoxin level <=0.07 EU/mg)) was purchased from Lifecore Biomedical (Chaska, MN, USA). Immunization studies, ELISA and cell culturing were performed by using endotoxin-free ovalbumin (OVA) (endotoxin level < 1 EU/mg) from Invivogen (Toulouse, France). PBS pH 7.4 for hMN injections was obtained from B. Braun, Melsungen, Germany. For cell culture, Ca 2+ -and Mg 2+ -free phosphate-buffered saline (PBS), Iscove's Modified Dulbecco's Medium (IMDM), Roswell Park Memorial Institute Medium (RPMI 1640), penicillin/streptomycin and l-glutamine were purchased from Lonza (Basel, Switzerland). Fetal calf serum (FCS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from GE Healthcare (Little Chalfont, UK) and PeptroTech (London, UK) respectively. Lipopolysaccharide (LPS) extracted from Salmonella typhosa, CFSE and βmercaptoethanol were purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands). The antibodies CD25-APC (PC 61.5), CD69-PE (H1.2F3), Thy1.2-PE-Cy7 (53-2.1), CD4-eFluor450 (GK1.5), Vα2 TCR-PE (B20.1) and fixable viability dye-eFluor780 were purchased from eBioscience (Thermofisher Scientific, MA, USA). For dissolving microneedle fabrication, 10 mM PB (7.7 mM Na2HPO4, 2.3 mM NaH2PO4, pH 7.4) was prepared in the laboratory. Vinylpolysiloxanes A-silicone (Elite Double 32a Normal) and the two-component epoxy glue (Bison, Goes, The Netherlands) were obtained from The Zhermack Group (Badia Polesine, Italy) and Bison International B.V. (Goes, The Netherlands), respectively. Polydimethylsiloxane (PDMS, Sylgard 184) was purchased from Dow Corning (Midland, MI, USA). Solid silicon MNs, obtained through a potassium hydroxide wet-etching process (31) , were kindly provided by the Tyndall National Institute (Cork, Ireland). All the chemicals were of analytical grade and Milli-Q water (18 MΩ/cm, Millipore Co.) was used for the preparation of all solutions.
Animals Immunization studies were performed using female BALB/c (H2d), 8-11 weeks old (Charles River, Maastricht, The Netherlands) and randomly assigned to groups of 8. The studies were approved by the ethical committee on animal experiments of Leiden University (License number (14176). For in vitro T-cell activation studies, C57BL/6 and OT-II transgenic mice on a C57BL/6 background were purchased from Jackson Laboratory (CA, USA), bred in-house under standard laboratory conditions, and provided with food and water ad libitum. All animal work was performed in compliance with the Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Experiments were approved by the Ethics Committee for Animal Experiments of Leiden University (CCD number AVD106002017887).
Hollow microneedles Hollow microneedles (hMNs) were prepared as described previously (32, 33) (a representative image of a hMN is reported in the Supplementary data, Figure S1 ). Briefly, polyimide-coated fused silica capillaries (Polymicro, Phoenix AZ, 375 μm outer diameter, 50 μm inner diameter) were filled with silicone oil in a vacuum oven (100 C) overnight. Subsequently, the capillaries were etched during 4 h in >= 48% hydrofluoric acid and the polyimide coating was removed from the ends of the capillaries by diving them into heated (250 C) sulfuric acid for 5 min.
Immunization studies BALB/c mice were anesthetized by intraperitoneal injection of 150 mg/kg ketamine and 10 mg/kg xylazine and the injection site was shaved (approximately 4 cm 2 ). The same day, mice were immunized by intradermal hMN injection (120 μm injection depth) of 10 μl with 0.31 μg OVA with 3.1 μg HA for each HA-MW dissolved in PBS pH 7.4. Intradermal hMN injection of each HA-MWs without OVA and PBS were included as control. For controlled depth intradermal microinjections, a hollow-microneedle applicator was used as reported previously (32) . Immunizations were performed at day 1 (prime), day 22 (boost) and day 43 (2nd boost). Prior to each immunization, a blood sample was collected from the tail vein. At day 63, the blood sample was collected from the femoral artery and all mice were sacrificed. Serum was isolated from the samples and stored at -80C.
Determination of OVA-specific IgG antibodies OVA-specific antibodies were analysed by a sandwich enzyme-linked immunosorbent assay (ELISA) as described earlier (10) . Briefly, well-plates were coated with OVA for 1.5 h at 37 C and then blocked with bovine serum albumin (BSA) (Sigma-Aldrich, Zwijndrecht, the Netherlands). After the blocking, three-fold serial dilutions of serum were applied to the plates and incubated for 1.5 h at 37 C. Then, the plates were incubated with horseradish peroxidase-conjugated goat antibodies against IgG total, IgG1 and IgG2a (Southern Biotech, Birmingham, AL, USA) for 1 h at 37 C. Finally, 1-step TM ultra 3,3′,5,5′tetramethylbenzidine (TMB) (Thermo-Fischer Scientific, Waltham, USA) was used as substrate and sulfuric acid (H2SO4) (95-98%) (JT Baker, Deventer, The Netherlands) was added to stop the reaction. The absorbance was measured at 450 nm on a Tecan Infinite M1000 plate reader (Männedorf, Switzerland) and the antibody titers were determined as the log10 value of the mid-point dilution of a complete s-shaped absorbance-log dilution curve of the diluted serum level.
Bone marrow-derived dendritic cells (BMDCs) To examine T-cell activation in vitro, first bone marrow was isolated from the tibias and femurs of C57BL/6 female mouse. A single-cell suspension of bone marrow cells was obtained by using a 70 μm cell strainer (Greiner Bio-One B.V., Alphen aan den Rijn, NL). The cells were cultured in IMDM medium supplemented with 2 mM l-glutamine, 8% (v/v) FCS, 100 U/mL penicillin/streptomycin and 50 μM β-mercaptoethanol at 37 C and 5% CO2 in 95 mm Petri dishes (Greiner Bio-One B.V., Alphen aan den Rijn, NL) and 20 ng/mL GM-CSF for 10 days. Medium was refreshed every other day. On day 11, the BMDCs were harvested from the petri dish and distributed into 96-well plates (100 μl/well, 10 000 cells/well). Then, 100 μl/well of formulations consisting of: i) 5 μg OVA, ii) 5 μg OVA mixed with 50 μg HA (per each molecular weight) or iii) 50 μg HA (per each molecular weight) were added to the wells. OVA (5 μg/well) + LPS (100 ng/well) or LPS (100 ng-well) were added as positive control; medium was included as negative control. The BMDCs were exposed to the formulations overnight at 37 C and 5% CO2 and subsequently OVA-specific CD4 + T-cells were transferred on BMDCs in co-culture experiments (see 2.6).
CD4 + T-cell activation by antigen loaded BMDC OT-II (OVA-specific CD4 + ) T-cells were obtained from the spleen of OT-II transgenic C57BL/6 mouse. Single cell suspensions were obtained by forcing the spleens through a 70 μm strainer. After erythrocyte depletion with lysis-buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA; pH 7.3), staining with CFSE was performed. Briefly, cells were suspended in PBS containing 1 μM CFSE and incubate 10 min at room temperature. CFSE was neutralized by FCS addition and the cells were washed with RPMI to remove excess of CFSE. At this point, the percentage of CD4 + /Valpha2 + cells was determined by flow cytometry (BD FACSCanto-II, San Jose, CA). OT-II cells were transferred on BMDCs previously exposed to the formulations (50 000 cells/well). On day 15, the cell surfaces were stained by incubating the cells with fixable viability dye-eFluor780 (1:1000) and fluorescently labelled antibodies specific for: Thy1.2-PE-Cy7 (1:500), CD25-APC (1:500), CD69-PE (1:500) and CD4-eFluor450 (1:500) for 30 min (100 μl/well) at 4 C. After 30 min, the excess antibodies were washed by using FACS buffer. The cells were incubated with fixation and permeabilization solution (BD Biosciences) for 10 min at 4 C. Finally, the cells were washed with FACS buffer and analysed by flow cytometry (BD FACSCanto-II, San Jose, CA). The data were analysed by using FlowJo software.
Fabrication of dissolving microneedles dMN arrays were prepared as described previously (10) . HA 10% (w/v) was dissolved in phosphate buffer ( Finally, the arrays were removed from the PDMS mold and inspected for shape and sharpness by light microscopy (Stemi 2000-C, Carl Zeiss Microscopy GmbH, Göttingen, Germany).
Human skin Human abdomen skin was obtained from a local hospital within 24 hours after cosmetic surgery according to the declaration of Helsinki. The fat excess was removed with a scalpel and the skin was stored at -80C. Before use, the skin was thawed at 37C for 1 h in a humid petri dish and stretched with pins on parafilm-covered styrofoam. Before starting the experiment, the skin was cleaned with Milli-Q and 70% ethanol.
Penetration of microneedles in ex vivo human skin dMN arrays (n=3 per HA-MW) were applied onto the skin by impact velocity, as described elsewhere (10, 34) , by using an impact insertion applicator with a constant velocity of 0.54 m/s (Leiden University -applicator with uPRAX controller version 0.3). The dMNs were kept in the skin for 18 seconds and withdrawn. Then, the pierced skin was treated with trypan blue, as previously described (35) and stratum corneum was removed by tape stripping. Finally, the blue spots were visualized using a light microscope (Axioskop and Stemi 2000-C, Carl Zeiss Microscopy GmbH, Göttingen, Germany) (a representative image of pierced skin is reported in the Supplementary data, Figure S2 ) and the penetration efficiency per array was calculated by dividing the number of blue spots by the number of dMNs on the array (16) (Equation 1 ).
Penetration efficiency = number of blue spots 16 x 100 (Eqn. 1)
Dissolution of microneedles in ex vivo human skin A dMN array (n=7) was applied on the skin as described in section 2.10 and was kept in the skin for 1 min, 5 min, 10 min or 20 min. The microneedle length before and after dissolution was determined with a light microscope (Axioskop and Stemi 2000-C, Carl Zeiss Microscopy GmbH, Göttingen, Germany) equipped with a digital camera (Axiocam ICc 5, Carl Zeiss). In order to obtain the dMN dissolved volume for each insertion period, dissolved microneedle length was measured by ZEN 2012 blue edition software (Carl Zeiss Microscopy GmbH) and the dissolved MN volumes were calculated as reported in our previous study in supplementary Figure 3 (10) .
Statistics IgG titers and T-cell response were analysed using one-way ANOVA with respectively Bonferroni and Tukey's post-test suitable in the software Prism (Graphpad, San Diego, USA). A p-value less than 0.05 was considered to be significant. Microneedle penetration efficiency was analysed by Kruskal-Wallis test with Dunn's multiple comparison test (p < 0.05). The remaining dMN length after dissolution at different time points was analysed by two-way ANOVA with a Tukey's post-test (p < 0.05).
Results
Immunization studies The liquid formulations of OVA alone or mixed with different HA-MWs was intradermally injected by using hollow microneedles. During injection no problems with clogging or leakage were observed. The formation of a blister on the site of the injection indicated a successful intradermal delivery of the formulation. No adverse effects were observed. The OVA-specific total IgG titers increased after each immunization (Figure 1A-C ). The presence of HA, regardless of the molecular weight, did not increase the total IgG response compared to the injection of OVA only. After the first boost, a higher OVA specific IgG response in the mice immunized with OVA-HA 4.8 kDa was observed compared to the mice injected with OVA-HA 150 kDa (p<0.01). However, both OVA-HA 4.8 kDa and OVA-HA
Analysis of CD4 + T-cell activation in vitro To determine whether the presence of different HA-MW, with or without OVA, affect celllar responses, OT-II T-cell (OVA-specific CD4 + cells) activation studies were performed in vitro. As expected, addition of OVA induces OT-II proliferation, which is enhanced in the presence of LPS (Figure 2 ). The co-exposure of cells to OVA and HA however, regardless of the molecular weight, did not increase OT-II proliferation compared to OVA alone. In line with the antibody responses, this suggests that none of the tested HA polymer provided a measurable adjuvant effect.
Dissolving microneedle fabrication The immunological analysis of the various HA polymers, suggests a preference for small MW polymer, as the high MW polymer show reduced antibody titers. Next we determined whether the MW of HA affected the fabrication of dissolving microneedles and their dissolution in the skin. Fabrication of dissolving microneedles was possible for the HA-MWs 150 kDa, 20 kDa and 4.8 kDa. The 10% (w/v) concentration of MW 1.8 MDa HA led to gel formation preventing the fabrication of microneedles. For the HA-MW 4.8 kDa, the fabrication of dissolving microneedles with a final step of drying at 37C, as performed for the 150 kDa and 20 kDa HA, led to ruptures on the microneedle arrays surface (data not shown). Thus, the drying was performed at room temperature, resulting in intact microneedle arrays.
Penetration and dissolution of microneedles in ex vivo human skin In order to assess whether the molecular weight of HA had an effect on the capability of microneedles to pierce and dissolve into the skin, first their ability to pierce ex vivo human skin was tested. Bright field analysis showed sharp microneedles regardless of the HA-MW (Figure 3A ).The penetration efficiency of microneedles into the skin was not affected by the molecular weight of the HA. The 150 kDa microneedles showed a penetration efficiency of 96 ± 7%; 20 kDa and 4.8 kDa HA microneedles showed a penetration efficiency of 98 ± 4%, (mean ± SD, n=3). Dissolution studies showed a gradual dissolution in time irrespective of the HA-MW (Figure 3A ). However, while 20 kDa and 4.8 kDa HA dMNs completely dissolved within 20 minutes application in the skin (9 7± 2% and 100 ± 0% dissolved volumes respectively), the 150 kDa HA dMNs reached only 80% dissolved volume after the same application time (Figure 3A ). No differences in dissolved volumes between 20 kDa and 4.8 kDa HA microneedles at each application time were observed (Figure 3B ), however the dissolved volume of the 150 kDa HA microneedles was significantly reduced at each time point compared to the other HA-MW microneedles. This resulted in a complete dissolution of the 20 kDa and 4.8 kDa HA microneedles after 10 minutes application, while the 150 kDa HA microneedles took 20 minutes to almost completely dissolve.
Discussion When choosing the HA polymer for the fabrication of dMNs, it is relevant to investigate potential immune modulating effects besides effects related to the dMN manufacturing and physicochemical characteristics of dMNs such as capability to pierce the skin and dissolve quickly. Although LMW-HA can have inflammatory properties in contrast to HMW-HA (19, 20, 22, 36, 37) , to our knowledge, the role of HA-MW on the antibody response has not been reported yet. To this end, in the present study HA ranging from 4.8 kDa to 1.8 MDa mixed with OVA was injected intradermally in mice to assess the antibody response evoked. We did not observe an increase in antibody titers by adjuvanting with LMW-HA; however we did observe that HMW-HA (1.8 MDa) reduced the antibody level after the second boost compared to OVA only and LMW-HA 4.8 kDa HA mixed with OVA. This may be explained by reports that HMW-HA exerts an anti-inflammatory role reducing the side effects of vaccines (38, 39) and displays immunosuppressive properties (40) (41) (42) . To this end, it has been reported that HMW-HA up-regulates the transcription factor FOXP3 on regulatory T-cells (Treg) (e.g. CD4 + CD25 + ) (42) involved in the regulatory mechanism of autoantibody production (43) and likely antibody production. Although we did not investigate Treg activation in this study, they provide a potential mechanism regarding the lack of immunogenicity of HMW-HA. The role of what in literature is defined as LMW-HA has been extensively investigated suggesting a pro-inflammatory effect in vitro: 4-6 oligosaccharides HA induced cytokine synthesis in dendritic cells (19, 20) ; HA < 250 kDa induced inflammatory cytokines levels (36, (44) (45) (46) (47) (48) ; HA <= 800 kDa led to activation of macrophages (37) . In this study, the effect of HA-MW was examined on the activation of CD4 + T-cells. Although this is an in vitro system, it has shown to be predictive in the sense that formulations that induce strong in vitro response of OT-II cells also outperformed other formulations in vivo (49, 50) . Conversely to what has been reported in literature, changes in HA-MW did not influence the proliferation and activation of CD4 + T-cells in vitro. Considerations justifying the referred controversial effects of HA preparations may be related to i) MW of HA that is not always measured accurately or is not homogeneous in the same HA population, for this reason an effect may be attributed to a minor population, ii) the presence of minor contaminants even in highly purified HA and iii) the conformational diversity of HA highly dependent on pH, temperature, salt concentration and specific cations (48) . Furthermore, the effects of HA-MW seems to be cell-specific and depending on the HA medium concentration (26, 48) . However, in literature there is not an optimal HA concentration reported and often the same concentration of different HA-MWs may have opposite effects on CD4 + cellular response (42) . This lack of data should be addressed in future investigations, i.e. i) HAs in a wide range of concentrations with a dose/response on specific cell types and ii) cytokines produced by DCs and CD4 + Tcells after exposure to the HA formulations, to obtain additional information on the regulatory effect of HA. Due to the absence of intrinsic adjuvant properties of HA, regardless of the molecular weight, all the HA-MWs were tested for potential effects on the manufacturing of dMNs and their capability to penetrate the skin and quickly dissolve. As reported in literature, a factor influencing the dMN dissolution time in the skin is the HA concentration (10, 51) . However, to our knowledge, the role of the HA-MW on the dMN dissolution in the skin is reported in the present study for the first time highlighting the novelty of this study. HA is being used in experimental drug delivery systems. The lack of immune activation of HA, regardless MW may be used to taylor drug release kinetics. However, although we did not show immunological effects, using low MW HA, may introduce safety concerns, since low MW fragments (oligomers) are associated with inflammatory responses (52) . Difficulties or impossibility in dMN fabrication when choosing too low (4.8 kDa) or high (1.8 MDa) HA-MWs, respectively, identified an optimal HA-MW in the middle range (20 kDa and 150 kDa). The 4.8 kDa HA could only be formulated into dMN by drying at room temperature to avoid ruptures of the dMN arrays. Although drying at low temperature (room temperature) can preserve the antigen stability, it may lead to long processing time not compatible with scalability in fabrication and to a higher residual moisture content in dMN than drying at high temperature, potentially resulting in decrease in antigen stability. The HA-MW 1.8 MDa formed a gel, due to the high viscosity, preventing further processing into dMNs. Furthermore, increase in HA-MW led to prolonged application time for a complete dMN dissolution into the skin. 10 mM, pH 7.4) and stored overnight. The next day, HA solution was poured in each well of the polydimethylsiloxane mold (PDMS, Sylgard 184, Dow Corning, Midland, MI, USA) to prepare dMN arrays (4 by 4 dMNs per array of 300 μm length and 100 μm base diameter). After several steps of vacuum and centrifuge, the 20 kDa and 150 kDa HA arrays were oven-dried overnight at 37C. As this procedure result in fragile dMN arrays when using 4.8 kDa HA, these dMN arrays were dried at room temperature overnight. The next day, a backplate was produced by pouring a mixture of vinylpolysiloxane base and catalyst (in a 1:1 ratio) (Elite Double 32a Normal, Zhermack Group, Badia Polesine, Italy) and subsequently a two-component glue solution (Bison International B.V., Goes, The Netherlands) onto each array and left curing.
1. 8 8 MDa resulted in equal responses compared to the OVA only group. After the second boost, the group immunized with OVA-HA 1.8 MDa showed lower OVA-specific total IgG titers than OVA only and OVA-HA 4.8 kDa, suggesting a detrimental effect on adding high molecular weight HA. Overall, the presence of high MW HA seemed to reduce the immunogenicity of the antigen while the presence of HAregardless of the molecular weight did not enhance the immune response compared with OVA alone. The IgG1 response (Figure1 D-F) followed the same trend as the total IgG response: the presence of HA in different molecular weights did not change the response compared to OVA alone after each immunization. Intradermal injection of OVA, with or without HA, resulted in undetectable OVA specific IgG2a responses (data not shown).
Figure 1 . 1 Figure 1. OVA-specific IgG total (A-C) and IgG1 (D-F) antibody titers measured in BALB/c mice on day 21 (A and D), day 42 (B and E) and day 63 (C and F). Bars represent mean ± SEM, n = 8. *p < 0.05, p < 0.01. OVA: ovalbumin; HA: hyaluronan.
Figure 2 . 2 Figure 2. OT-II activation in vitro. (A) percentage of dividing cells, (B) percentage of CD69 + /CD25 + cells. Bars represent mean ± SEM, n = 3. *p < 0.05, p < 0.01. OVA: ovalbumin; HA: hyaluronan; LPS: lipopolysaccharide; s.d.: significantly different.
Figure 3 . 3 Figure 3. dMN dissolution in ex vivo human skin. A) Representative bright field images (5x) of 10% (w/v) HA 4.8 kDa, 20 kDa and 150 kDa microneedle before application on the skin (0 min) and after 1, 5, 10 and 20 min dissolution in ex vivo human skin. Dissolved volumes are reported as mean ± SEM (n = 7). Scale bar 100 μm. B) Effect of time (1, 5, 10 and 20 min) on dissolution per each HA-MW dMN type (statistics in black) and effect of HA-MWs on
|
10.1101/2020.04.22.20075200
|
public-domain
| null | null |
openalex
|
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), a low prevalence area. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was tested using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10.90 ± 3.54% and 10.85 ± 2.11% in influent water and 3.29 ± 1.58% and 6.19 ± 1.00% in effluent water samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater waters of 5.29 log genomic copies/L on average. Moreover, we tested as negative all secondary and tertiary treated water samples, highlighting that current water disinfection treatments applied in the analyzed WWTP are able to remove SARS-CoV-2 RNA. This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that SARS-CoV-2 was circulating among the population even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.Introduction Coronaviruses (CoVs) are a family of viruses pathogenic for humans and animals associated to respiratory and gastro-intestinal infections. CoVs used to be considered as minor pathogens for humans as they were responsible of common cold or mild respiratory infections in immunocompetent people. Nonetheless, the emergence of novel and highly pathogenic zoonotic diseases caused by CoVs such as Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and most recently SARS-CoV-2 arises questions to be addressed to guide public health response. CoVs are mainly transmitted through respiratory droplets. However, as for SARS and MERS, SARS-CoV-2 RNA has been detected in stool samples from patients suffering COVID-19 and from asymptomatic carriers (1) (2) (3) (4) (5) . The duration of viral shedding has been observed to vary among patients with means of 14-21 days (6, 7) , as well as the magnitude of shedding varies from 10 2 up to 10 8 RNA copies per gram (1, 2, 8) . Infectious viruses deriving from fecal specimen have been cultured in Vero E6 cells and observed by electron microscopy (9) . In addition, gastric, duodenal, and rectal epithelial cells are infected by SARS-CoV-2 and the release of the infectious virions to the gastrointestinal tract supports the possible fecal-oral transmission route (10) . Even though the possibility of faecal-oral transmission has been hypothesized, the role of secretions in the spreading of the disease is not clarified yet (6, 7, 9, 11) . Wastewater monitoring has been a successful strategy pursued to track chemical and biological markers of human activity including illicit drugs consumption, pharmaceuticals use/abuse, water pollution, and occurrence of antimicrobial resistance genes (12) (13) (14) (15) . Viral diseases have been also surveilled by the detection of genetic material into wastewater as for enteric viruses (16) (17) (18) , re-emerging zoonotic hepatitis E virus (19, 20) , and poliovirus during the global eradication programme (21) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint Currently, various studies detected SARS-CoV-2 RNA in wastewater worldwide (22) (23) (24) (25) (26) , and wastewater testing has been suggested as a non-invasive early-warning tool for monitoring the status and trend of COVID-19 infection and as an instrument for tuning public health response (27) (28) (29) . Under current circumstance, this environmental surveillance could be implemented in wastewater treatment plants as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown. Here, we report the first detection of SARS-CoV-2 RNA in untreated wastewater samples in Spain collected from six different wastewater treatment plants (WWTPs) in Murcia, the lowest prevalence area in Iberian Peninsula. Additionally, the efficacy of the secondary and tertiary treatments implemented in the WWPTs against SARS-CoV-2 has been confirmed. The outcomes of the environmental surveillance reflect the epidemiological data in a low COVID-19 diagnosed cases setting, thus supporting the need of developing and implementing advanced models for wastewater-based epidemiology (WBE). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint
Material and methods
Sampling sites and samples collection Influent and effluent water samples were collected from six WWTPs located in the main cities of the Region of Murcia, Spain (Figure 1 ). Technical data on WWTPs are provided in Table 1 . A total of 42 influent, and 18 secondary and 12 tertiary treated effluent water samples were collected from 12 March to 14 April 2020 and investigated for the occurrence of SARS-CoV-2 RNA. Collected samples were transferred on ice to the laboratory and concentrated on the same day of sampling or the day after.
Wastewater and effluent water concentration The porcine epidemic diarrhea virus (PEDV) strain CV777 and the mengovirus (MgV) vMC0 (CECT 100000) were preliminary used to evaluate the aluminum hydroxide adsorption-precipitation method previously described for concentrating enteric viruses from wastewater and effluent water (30, 31) . In brief, 200 mL of biobanked influent (n=2) and effluent water samples (n=2) were artificially inoculated with PEDV and MgV. Then pH was adjusted to 6.0 and Al(OH)3 precipitate formed by adding 1 part 0.9N AlCl3 (Acros organics, Geel, Belgium) solution to 100 parts of sample. The pH was readjusted to 6.0 and sample mixed using an orbital shaker at 150 rpm for 15 min at room temperature. Then, viruses were concentrated by centrifugation at 1,700 × g for 20 min. The pellet was resuspended in 10 mL of 3% beef extract pH 7.4, and samples were shaken for 10 min at 150 rpm. Concentrate was recovered by centrifugation at 1,900 × g for 30 min and pellet resuspended in 1 mL of PBS. All wastewater and effluent water samples included in this study were processed as described and MgV (5 log PCRU) was spiked as process control.
Viral extraction, detection and quantification Viral RNA was extracted from concentrates using the NucleoSpin RNA virus kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer's instructions with some modifications. Briefly, 150 μL of the concentrated sample was mixed with 25 μL of Plant RNA Isolation Aid (Thermo Fisher Scientific, Vilnius, Lithuania) and 600 μL of lysis buffer from the NucleoSpin virus kit and subjected to pulse-vortexing for 1 min. Then, the homogenate was centrifuged for 5 min at 10,000 × g to remove the debris. The supernatant was subsequently processed according to the manufacturer's instructions. Viral RNA was detected by TaqMan real-time RT-PCR (RT-qPCR) on LightCycler 480 instrument (Roche Diagnostics, Germany). MgV RNA was detected by using UltraSense One-Step kit (Invitrogen, SA, US) and the RT-qPCR assay as in ISO 15216-1:2017 (32, 33) . Undiluted and ten-fold diluted MgV RNA was tested to check for RT-qPCR inhibitors. PEDV RNA was detected by using PrimeScript One Step RT-PCR Kit (Takara Bio, USA) and the TaqMan RT-qPCR assay described by (34) . SARS-CoV-2 RNA was detected by using PrimeScript One Step RT-PCR Kit and the RT-qPCR diagnostic panel assays validated by the US Centers for Disease Control and Prevention (35) . The first version of the kit with three sets of oligonucleotide primers and probes was used to target three different SARS-CoV-2 regions of the nucleocapsid (N) gene. The sets of primer and probes (2019-nCoV RUO Kit) as well as the positive control (2019-nCoV_N_Positive Control) were provided by IDT (Integrated DNA Technologies, Leuven, Belgium). Each RNA was analyzed in duplicate and every RT-qPCR assay included negative (nuclease-free water) and positive controls. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint Biobanked samples (n=4) collected in October 2019, before the first COVID-19 case was documented, were used as relevant negative control to exclude false positive reactions. SARS-CoV-2 RNA was quantified as genome copies (gc) by plotting the quantification cycles (Ct) to an external standard curve built with 10-fold serial dilution of a quantified plasmid control (IDT). MgV and PEDV RNA were quantified by plotting the Cts to an external standard curve generated by serial end-point dilution method. MgV recovery rates were calculated and used as quality assurance parameters according to ISO 15216-1:2017 (33).
Results and Discussion
Performance of the concentration method The aluminum hydroxide adsorption-precipitation method was tested by spiking influent and effluent samples with MgV and PEDV. On average, MgV was recovered at ranges of 10.85 ± 2.11% in influent and 6.19 ± 1.00% in effluent water. PEDV was recovered at ranges of 10.90 ± 3.54% in influent and 3.29 ± 1.58% in effluent water. These results are in line with the MgV recoveries reported for enteric viruses concentration in water samples by the same aluminum-based method (19, 30) and higher than the 1% as the quality assurance parameter indicated for bottled water into ISO 15216-1:2017 (33) . Similarly, MgV was successfully used as recovery control for hepatitis E virus concentration from influent and effluent water samples (5-13%) by applying a polyethylene glycol (PEG) precipitation method (20) . A similar PEG-based protocol was recently used to recover SARS-CoV-2 from wastewater, although recovery control was not included in the study (23) . Moreover, filtration through 10 kDa Centricon Plus-70 centrifugal device successfully recovered SARS-CoV-2 in wastewater with recovery efficiencies of F-specific RNA All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint phages of 73% (22) . However, concentration by electropositive membrane should be further evaluated given a SARS-CoV recovery from wastewater of 1% (36) . Rigorous limits of detection should be established by spiking SARS-CoV-2 cell-culture adapted strain or positive COVID-19 fecal samples in influent and effluent wastewater samples to be concentrated following the aluminum hydroxide adsorption-precipitation method. Nonetheless, the need of a BSL3 laboratory facility to handle SARS-CoV-2 represents the main limitation of this experiment.
SARS-CoV-2 titers in wastewater and effluent water A total of 42 influent, and 18 secondary and 12 tertiary treated effluent water samples A relevant number of influent water samples (12%) showed Ct ranging between 37 and 40, even though lower Ct of 34-37 were observed (29%). In all samples, MgV recoveries were above 1% (10.05 ± 14.10%). Detection of SARS-CoV-2 RNA in influent water has been reported worldwide (22) (23) (24) 26) , and only one study tested treated wastewater that resulted positive (Paris) (25) . On average, SARS-CoV-2 RNA titers of 5.15 ± 0.25, 5.53 ± 0.24, and 5.49 ± 0.27 log gc/L were quantified in wastewater by using N1, N2 and N3 primer/probe mixes, respectively. Titer of 4 and 5 to more than 6 log gc/L have been reported in Massachusetts and France, respectively (23, 25) . We observed discrepancies among RT-qPCR N1, N2 and N3 assays for several water samples in agreement to a previous report (22) . This could be due to the different analytical sensitivity among the assays as well as the detection of possible false positive samples by RT-qPCR N3 in low concentrated samples (37, 38) . The latter possibility has been solved by excluding the N3 primers/probe set from the US CDC 2019-nCoV RT-qPCR diagnostic panel in its last revision (March, 30) (39) . In addition, a partial inhibitory effect of the matrix is not to be completely excluded despite the controls included in the assays. A more sensitive estimation of SARS-CoV-2 loads in wastewater should be studied by digital RT-qPCR (dRT-qPCR). dRT-qPCR could be used to quantify samples with low viral loads, even though it may not be the best practical and economically sustainable option for environmental surveillance. Even though the SARS-CoV-2 RNA detection in wastewater is functional for WBE purposes, the risk for human health associated to the water cycle is still under debate as infectivity of viral particles remain to be confirmed as well as its potential fecal-oral transmission. In spite of the high concentration of viral RNA in specimen and the evidence of gastrointestinal infection (10) , infectious viruses from stools have been isolated in one study (9) while another attempt resulted without success (2) . The potential transmission of SARS-CoV-2 via wastewater has not been proven (40, 41) and it seems unlikely given the poor viral stability in environmental conditions and its elevated sensitivity to disinfectants (26, 42, 43) .
Environmental surveillance Epidemiological data on COVID-19 in the Murcia Region have been retrieved from the publically available repository of the "Servicio de epidemiologia" of the "Consejería de Salud de la Región de Murcia" (available at http://www.murciasalud.es/principal.php) (Table 3 ) and plotted to the SARS-CoV-2 RNA mean loads as detected by three RT-qPCR assays (Figure 2 ). In general, RT-qPCR amplification signals have been detected in wastewaters when cases were diagnosed within the municipality. Positive wastewater samples have been detected with at least two out of three RT-qPCR assays in low prevalence municipalities as in Murcia (96 cases, 21.18 cases per 100,000 inhabitants), Cartagena (36 cases, 16.76) and Molina de Segura (12 cases, 16.69). Of note, positive wastewater samples were detected 12-16 days before COVID-19 cases were declared in Lorca, Cieza and Totana municipalities. A similar study conducted in Paris (France) demonstrated the detection of viral genome before the exponential phase of the epidemic (25) . However, our results indicate that SARS-CoV-2 can be detected weeks before the first confirmed case. The early detection of SARS-CoV-2 RNA in wastewater could have alerted about the imminent danger, giving a valuate time to the managers to coordinate and implement actions to slow the spread of the disease. Therefore, our outcomes support that WBE could be used as an early warning tool to monitor the status and the trend of COVID-19 infection within a community. On the other hand, we believe that this environmental surveillance could be used as an instrument to drive the right decisions to reduce the risk of lifting restrictions too early. In fact, a very important question is to determine what needs to be implemented to have reliable data to reduce the risk of a "second wave". Massive population tests are the first choice, but in their absence, wastewater monitorization of SARS-CoV-2 RNA can give a All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint reliable picture of the current situation. Our wastewater data do not quantitatively resemble the prevalence of COVID-19 confirmed cases. To this end, a quantitative model that includes and corrects all the variables affecting these wastewater surveillance data would be useful for a better interpretation. For instance, not all COVID-19 positive patients excrete SARS-CoV-2 RNA in faeces, and when it occurs, the titers and the duration of shedding vary among individuals and across time (1, 2, 5, 7). On the other hand, the real number of positive cases within the Murcia Region remains unknown because of the large number of mild or asymptomatic carriers that have not been included in epidemiological statistics. These aspects together with environmental variables (e.g., rainfall events, temperature) increase the uncertainties linked to the correlation between SARS-CoV-2 RNA detection in wastewater samples and the prevalence of COVID-19 that could be explored by using complex models. In conclusions, wastewater surveillance and WBE may represent a complementary approach to estimate the presence and even the prevalence of COVID-19 in communities. This represents an effective tool that needs to be further explored in order to direct public health response, especially in cases of limited capacity for clinical testing. were collected from 12 March to 14 April 2020 and investigated for the occurrence of SARS-CoV-2 RNA. Samples were considered positive for Ct below 40 (as in Medema et al., 2020 and F. Wu et al., 2020) and titrated by using the quantified plasmid control for each of the RT-qPCR targets. The 85.7% (36 positive samples out of 42) influent samples were tested positive for at least one SARS-CoV-2 RT-qPCR target. None (0 out of 30) of the secondary and tertiary effluent samples tested positive for any of the SARS-CoV-2 RT-qPCR target (Figure 2).
Figure 1 . 1 Figure 1. Maps of the sampling location. Symbols represents WWTPs and are sized
Figure 2 . 2 Figure 2. Mean amplification cycles of SARS-CoV-2 RNA in influent, secondary and
Figure 3 . 3 Figure 3. COVID-19 prevalence and SARS-CoV-2 RNA loads in wastewater per
Table 1 . 1 Operating characteristics of WWTPs.
|
10.1056/nejm189203101261001
|
public-domain
| null | null |
openalex
|
The following two cases of cerebral disease caused by the grippe are of interest because they illustrate the profound effect of the poison of the disease upon the central nervous system. One was a case of confu- sional insanity, the other was of doubtful nature. As a rule, confusional insanity ends favorably, but this case illustrates the possibility of fatal termination. Although the subject had suffered frpm chronic bron- chitis for many years, his physical condition otherwise was good up to the timo of his infection with the grippe, and therefore I think his previous physical condition contributed little towards tho fatal result, although from reading, merely, an account of the case, it might seem otherwise. Attention has often beeu called, and of lato particularly by Althaus and Mills,1 to the affinity of the poison of influenza for the nervous system. Although Althaus, it seems to me, goes too far in relegating the inflammation of the mucous membranes to a place of secondary importance and in considering the disease as an essentially nervous fever, still it. does not seem to tue that the action of the poison upon the nervous system in cases of the bronchitic type is regarded in practice as having tho importance that should be attached to it. Symptoms and physical conditions are ascribed to gen- eral weakness and exhaustion, which are in reality due to what might be better called a paresis of the nerve centres. In other words, the so-called exhaustion of grippe is not an exhaustion in tho true sense of the word, but is really the expression of a true neurosis which is due to the toxic or paralytic effect of the poison on the nerve centres, just as the paralysis of diphtheria is toxic aud not exhaustive. When both pulmonary and abdominal symptoms aro absent, the neurosis stuuds out prominently in the foreground and is plainly recognized ; but when these symptoms are present their more strikingly objective character over- shadows the neurosis which in consequence is apt to be overlooked or underestimated.2 A prolonged con- valescence with its train of nervous symptoms and even death then come not unfrequently as a surprise. The fatal result, so often unexpected, is fully as much, in my judgment, due to this neurosis as to the bronchitis. The latter, if occurring by itself, would not be sufficient to cause death. The acceptance of these views must affect the after- treatment and the prognosis. Cask I. The patient, a man sixty-six years of age, and a sufferer for the past thirty-five years with chronic bronchitis, was taken December 19th last with symp- toms of an acute exacerbation. As the patient had had several such attacks in the past it was impossible, at first, to say whether or not the acute bronchitis was due to the grippe or was simply an increase of the old trouble. There were no symptoms, in the beginning, to throw light on the etiology. Later, it became evi- dent that his symptoms were caused by grippe. The fever was moderate and lasted about a week or ten days ; the highest point reached was 102.f>, although it should bo said the temperature might have beeu modified by the phenacetine, which was given in mod- erate doses. The bronchitis was very severe. Very large quan- tities of muco-purulent sputa were raised in the twenty- four hours ; and the patient suffered from intense dyspnoea, which prevented his lying down in bed. It should be said that repeated examinations of his lungs during the past year had shown the existence of marked emphysema, bronchiectasies, and abundant coarse and fine moist râles throughout the lungs. The apices were particularly affected, and the right one in particular gave physical signs of phthisis. Examina- tion of the sputa had failed, however, to show the ex- istence of bacilli, and while the objective signs sinii- lated those of phthisis, the symptoms were all those of bronchitis. With the exception of great dyspnoea on exertion, and cough, particularly in the mornings, when the patient raised considerable quantities of muco- purulent expectoration, he had enjoyed fair health. His physical appearance had always been good and was in no way suggestive of phthisis. Dr. F. C. Shattuck, who saw the case with me in consultation, confirmed this view of the lungs, excepting that he considered there was a fibroid condition also present. After the first week of tho attack of grippe, the bronchitis gradually and steadily improved until, at the end of his illness, it had subsided to its previous condi- tion. The cough was moderate, troubling him but little, and the expectoration was comparatively slight. The sputa were again examined for bacilli, with nega- tive results. The interest of the case centres in the nervous symptoms. During the first week the patient suffered from marked depression, so marked, indeed, as to almost amount to melancholia. However, as it was known that he always became very much depressed when ill, it did not excite very much attention. During the next week, a stuporous condition developed. The pa- tient frequently fell, if not into a sleep, at least into a somnolent condition while talking, and it was with difficulty that his attention could be held for more thau a few minutes at a time. During tho third week his mental condition changed again ; the stupor disappeared ; the melancholia gave placo to a condition approaching exaltation. He appeared to be in abnor- mally good spirits, and to have entirely lost all appre- hension on account of his condition. At the same time a peculiar kind of delirium now developed resem- bling in many respects that of delirium tremens. At first, it was limited to bad dreams; with these the pa- tient was tormented, particularly at night, but also if lie fell asleep in the daytime. By degrees the dreams persisted after he was waked up, and he seemed to find it difficult to throw them off. Then they persisted as false ideas and experiences during the waking state, and he became unable to distinguish between his dream life aud his real life. . By degrees hallucinations and illusions developed during the day; he saw people iu his room, and objects outside in the street, which did not exist, and a chain of delirious ideas took complete possession of his mind. He found difficulty at times in expressing himself intelligently. Wrong words were 1 An oxeolleiit editorial in n hito number of this Journal has em- phasized this point. > In tho Journal of February 4th hist, two eases are roported of tho supposed etl'oct of Bulphonul in producing a reltipse (!) with nervous symptoms. It serins to me these are plainly eases of neurosis due to the grippe, mid Illustrate the point mudo In the text. interpolated in his sentences, and the words themselves slovenly pronounced. The mental confusion was marked in regard to his surroundings. He imagined himself in various and impossible situations. He recog- nized, however, everybody about him and the facts of his illness. In fact, his mental condition was, as has been said, very similar to that of delirium tremeus. The marked difference was, that while he was, and had been, during the whole attack tormented by bad dreams, and he required hypnotics every night, sleep was easily induced by moderate doses. Hyoscin, in doses of one one-hundred-and-fiftieth of a grain, gave quiet sleep during the greater part of the night. Examination of the urine showed the kidneys to be free from disease. Eight days before he died Dr. Jelly saw him with me in consultation. At that time there did not seem any reason for apprehending a fatal issue, but during the next few days his mental condition rapidly grew worse. An interesting point in the case, and one which, to my mind, bears upon the pathology of such cases, was the fact that while his mental condition was progres- sively growing worse, his physical condition was pro- gressively improving. The bronchitis was subsiding ; his dyspnoea was growing less ; his appetite improved so that he took large quantities of food, iu fact, more than he had been iu the habit of taking previous to his illness, and his strength increased to the extent that he was able to sit up in a chair for seven or eight hours a day. His pulse remained strong and full up to the last few days of his illness, aud ranged in the neighborhood of 90. When the hallucinations devel- oped he was put back to bed, aud he was treated witli large doses of cardiac and cerebral stimulants. His mental condition grew steadily worse, and he died January 25th, thirty-nine days after being taken sick. It was only during the last two or three days of his illness that he failed physically, aud on the last day he sank rapidly. Cash II. This case I saw in consultation with Dr. John G. Blake, who has kindly given me his notes iu the case. The patient, a man, forty years old, had always been more or less affected with chronic bronchitis of moderate intensity. He was also of moderately alcoholic habits, depending upon the use of stimulants in rather excessive quantities, although never, perhaps, intoxi- cated. He was attacked with grippe in January last, and attended by Dr. Blake. He was ill two weeks, the principal symptoms being those of bronchitis. At the end of two weeks the symptoms subsided, and he recovered strength sufficiently to be up and about the house and even to walk a short distance to Dr. Blake's office ; he still, however, continued weak. At the end of about five days severe neuralgic pains developed in the scalp ; these appear to have been in- tense, and the scalp became very sensitive to the touch. For two days, however, he appeared to be otherwise well, and talked rationally. He then became troubled at night with delirium, or dreams, which gradually per- sisted into the day. There had been no vomiting or convulsions. A delirious condition with hallucinations rapidly developed ; and when seen by me, iu consulta- tion, on the morning of the fourth day, his condition was as follows : He was in bed, but his muscular condition seemed to be strong; pulse, strong and full; not rapid, nor abnormally slow. He was in a stuporous condition, although it was evident that his mind was occupied with hallucinations. When disturbed, he played with the pillow and moved about in bed. There was no paralysis of any kind : he protruded his tongue, and answered questions. His condition gave the impres- sion of a meningitis. Insomnia had been marked. The temperature was not taken with the thermome- ter, but to the touch it did not appear to be raised. Dr. Blake says there never had been any suspicion of fever. Arrangement was made to transfer trie patient to the hospital ; but later some objection having been made, he did not go. " That evening his delirium," Dr. Blake says, " became even more marked and was of the muttering variety." He talked indistinctly aud incoherently. He could not be roused to answer in- telligently. The pulse continued full and strong. There was retention of urine and he required to be catheretized. The report of the night-watch was that he continued about the same until about 4 A. m. He then became more restless and-began to fail. He died about 7 a. M. Thus it will be seen that the chief and almost only symptom was delirium preceded by neu- ralgic pain iu the head. An autopsy was not allowed. The diagnosis lay between meningitis and some form of toxic cerebral disease duo to the poison of grippe. It was expected that after the patient's transfer to the hospital a more satisfactory study could be made, but his unexpected death prevented this.
REMARKS. The first of the above cases was plainly one of con- fusioual insanity, a name given by Wood, of Philadel- phia, to designate the form of mental disturbance often met with following acute fobrile diseases such as acute rheumatism, typhoid fever, grippe and even surgical operations. This form of brain disease has, of late, attracted much attention, both here and in Germany, and several monographs have appeared upon the sub- ject. A number of cases are to be found reported in the medical journals, Osier, for instance, has reported five cases, of which four followed typhoid. Wood, who has most exhaustively discussed it, considers that pathologically it is due to the exhaustion of the nerve cen- tres and not to the toxic effect of the specific poison of such diseases as above mentioned. He believes that its pathology is always the same, whether following a sur- gical operation, the puerperal condition or acute infec- tious diseases. These conditions induce a state of exhaustion which iu turn brings about one of mal- nutrition of the brain. It seems to me that this view may bo fairly questioned. It is undoubtedly true that mental disturbance may follow physical exhaustion, puro and simple, as, for example, when produced by starvation and exposure after shipwreck. Still, it seems to me that clinical evidence also points to the direct toxic action of the poison itself as the true cause in many, if not all, cases of acute infectious diseases. In the case just quoted, for example, it will be noticed that the first mental disturbance, namely, the melan- cholia developed during the few days of the disease before exhaustion could properly be said to have oc- curred : and more than this, instead of the physical condition of the patient deteriorating as the mental disturbance progressively increased, on the contrary, the physical strength steadily improved while the stu- por, hallucinations and other mental symptoms stead- ily grew worse. In fact, while the appetite, strength and the bronchitis were steadily improving, he was mentally failing. Nor was there at any time in the course of the disease evidence of weakened function of the vital organs beyond a temporary gastric disturbance shown by an exceedingly furred tongue, due partly to over-feeding. Taking theso facts into consideration, and taking also into consideration the fact that delirium iu grippe may be prominent on the first day without fever, bron- chitis or other local symptoms (one example of which I have seen), and that it may also occur early in gon- orrhoea] rheumatism before any exhaustion has devel- oped (an example of which I have also seen) I am dis- posed to consider the psychical disturbance as dependent on the direct toxic effect of the poison on the cerebral tissue. An analogous condition, and one beyond question, is tho mental disturbance observed in Bright's disease. This distinction is of some practical importance because if the weakness, or rather, as I believe it should be regarded, the paresis, the mental and other symptoms occurring during and following grippe are simply to be regarded as evidences of exhaustion, the treatment is simple and the prognosis will have a more favorable outlook ; but if these conditions are in reality toxic in their character, it is evident that the treatment of this nervous condition should be much more watchful aud serious and the prognosis more guarded. I am certain that the surprise often expressed at tho uuexpected fatal termination of many cases of grippe is due to the profound toxic effect of the poison on the nervous sys- tem not having been recognized. It is not a mere weakness that we have to do with but a roal paralysis of the nervous system. I am inclined to regard the great fatality in aged people as due only in part to the local lung complications, pure and simple, and at least iu an equal degree to this nervous poisoning often re- sulting in paralysis of the heart. It is true that con- fusional insanity in the great majority of cases has a favorable termination, but the above case illustrates its dangers. I am sorry that the sudden termination of the second case, above described, prevented more care- ful study of the conditions it presented. I think the diagnosis must remain iu doubt, but that it will lay between meningitis and toxic neurosis. Visiting Physician to the Boston City Hospital and the Children's Hospital. The following cases are reported more for their clinical interest than for making any precise deductions as to the amount of atropia or morphia which may produce death or be recovered from at any especial age in infancy or childhood. In fact, any such de- ductions must be based on an extremely large Dum- ber of cases to be of any definite value, for infants and children appear at the same age to vary considerably as to the amount of these drugs which they can absorb and eliminate without physiological or toxic symp- toms. A dose of belladonna may be taken without marked effect by an infant, and yet the same dose pro- duce toxic phenomena in au older child ; and in like manner we notice tho same want of uniformity in the effects produced by opium and its alkaloids. The ad- ministration of both belladonna and opium, however, plays so great a rôle in the treatment of diseases'of infancy and childhood that cases where any approach to toxic symptoms have occurred when these drugs have been used, should immediately bo put on record. In this way we shall, by degrees, learn to know what are the danger-signals aud what limits we should hesi- tate to pass beyond at a given age. These records will also be of use in encouraging us not to despair of saving the patient's life in cases where the amount of the drug taken seems very formidable. During my term of service at the City Hospital in 1891, a little girl, nine years old, was brought to my wards at 10 P. M. At about 7 P. si. on the same eve- ning, the child stated that she had drunk part of the contents of a bottle containing a solution of atropine, two grains to the ounce. An emetic of mustard and water was given to the child at 9 P. II., before coming to the hospital, and this had produced free vomiting. The child, on entrance to the ward, was partly uncon- scious ; pale; pulse 120, of good strength aud regular ; respiration regular, quiet aud about normal rate ; tem- perature 99; tongue coated and dry ; pupils widely dilated and not reacting. Couvulsive movements chiefly confined to the upper extremities occurred at times. The urine was of a normal color, acid, specific gravity 1020, no albumen. The case did not appear to require any treatment by drugs. On the following morning the child was comfortable with the exception of a slight headache. The pupils were still widely dilated. The urine was passed natu- rally. Three days later she was well and the pupils were reacting naturally. TJ)e sister of the child just spoken of was brought iuto the hospital at the same time aud with the same history and was placed in the wards of Dr. J. G. Blake. This little girl was four years old. She had been given an emetic two hours after drinking the atropine solution, and one hour before entering the hospital. The face was markedly flushed : the pupil of the left eye was fully dilated aud did not react to light. The right pupil, which was the seat of an old iritis, did not dilate ; the pulse was 160, regular, rather full and compressible ; respirations 28, regular and quiet ; temperature 100. Every few minutes the patient had mild, clouic convulsions, affecting chiefly the muscles of the arms, legs and face. They were of short duration. As the patient was in the stimulant stage of the poison, with convulsions, sho was given two minims of Magendie's solution, subcutaneously. After a short time the patient quieted down and went to sleep. By midnight the respirations had fallen to 22, the pulse to 130 (of good strength), and the patient was quiet. On the following morning the pulse was 92 and the respirations 18; the child was drowsy and fretful and the pupil still dilated. Three days later the child seemed well, and the pupil was gradually contracting. One-half to one grain of atropia has been considered a fatal dose for an adult so that the amount of atropia iu the solution (two grains), presumably swallowed by these two children would appear to be a serious dose to be disposed of. The probability is that they did not get a grain each ; but the bottle had only had a few drops taken out of it before they got possession of it, and was found empty after they both said they had drank some of its contents. The usual history, I be- lieve, of belladonna or atropia poisoning is that the patients recover ; and it is on this ground that the 1Read before the Obstetrical Society of Boston, January 9, 1892. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 12, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright 2010 Massachusetts Medical Society.
CASES OF ATROPINE AND OPIUM POISONING IN EARLY LIFE.1BY T. M. ROTCH, M.D.,
The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 12, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright 2010 Massachusetts Medical Society.
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 12, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright 2010 Massachusetts Medical Society.
|
10.4314/gjass.v22i1.5
|
cc-by
| null | null |
openalex
|
The broad objective of this study is the analysis of the long and short run effects of agricultural credit on cassava yield in Nigeria. The study applied descriptive statistics and autoregressive distributed lag (ARDL) econometric) approach to the secondary data collected from Central Bank of Nigeria Statistical Bulletin and FAOSTAT. The descriptive analysis shows that cassava output was increasing in Nigeria with decreasing trend in yield. It was revealed in the study that Roots and Tubers Expansion Programme (RTEP) has no significant effect on cassava production and yield. The result of the econometric analysis indicates that agricultural credit do not have significant long and short-run effects on cassava production and yield. This is because the real per capita agricultural credit is small. However, study indicates that fertilizer has long and short-run effects on cassava output but does not have a significant effect on cassava yield. This study also documented evidence of declined labour and land productivity on the long and short-run. The study reveals that ecessive rainfall can be detrimental to cassava production in the long and short-run. The study concludes that although, agricultural credit do not have significant long and short-run effects on cassava production and yield, it is positively correlated with agricultural inputs used in cassava production. The study recommended the need to address the decline in the real value of per capita credit by increasing the amount of credit given to the farmers. There is also the need to design more credit facilities suitable to the farmers. The current Anchor Borrowers programme should be designed to accommodate more resource-poor cassava farmers.BACKGROUND INFORMATION In order to encourage the agricultural and food production sector, the government over the years has put in place various policies and measures to boost food and agricultural production in Nigeria. Some of these programmes, projects and institutions are: Commodity and Marketing Board, National Accelerated Food Production Programme (NAFP), Operation Feed the Nation (OFN), National Seed Supply Services (NSS), Land Use Decree (LUD); Green Revolution (GR); National Agricultural Land Development Authority (NALDA), Agricultural Project Monitoring and Evaluation Unit (APMEU), Structural Adjustment Programme (SAP), the Presidential Initiative on cassava production and Agricultural Credit Guarantee Scheme Fund (ACGSF). ACGSF is one of the most sustained institutional agricultural credit schemes in Nigeria (Mafimisebi et al., 2008; Onuselogu, 2014; Adetiloye, 2012; Nwosu et al., 2010) . The Scheme was established to provide guarantee on loans granted by banks to farmers for agricultural production and agro-allied processing (Nwosu et al., 2010) . The ACGSF is available to provide succor to banks that lend to farmers under the programme (Mafimisebi et al., 2008) . It was established to help farmers who have little or no collateral to get loans from commercial banks by Central Bank of Nigeria (CBN) . It aimed at solving the problem of inadequate funding of farm operators by banks and to cushion these financial institutions against the effects of high risks associated with investments in farm enterprises as well as to raise the productivity and earnings from farm investments so that the incidence of loan repayment default among the farmers will be minimized. As agriculture is important to the Nigerian economy so is the root and tuber (which are mainly cassava and yam) crops sector in Nigeria. Tuber and root crops, including cassava, yam, potato and sweet potato are the most important food crops for human consumption in Nigeria (IITA, 2015) . These crops are cultivated in varied agro ecologies and production systems contributing to more than 240 million tonnes annually, covering up to 23 million hectares. The aggregate value of yam, cassava, potato and sweet potato surpasses all other staples grown in Nigeria (IITA, 2015) . FAOSTAT (2019) demonstrated that that roots and tubers constitute about 49% of total crops produced in Nigeria between 1981 and 2017, while cereal, coarse grains, fruits and vegetable constitute about 17%, 14%, 7% and 7% respectively. Olomola et al (2015) revealed the relative importance of root and tuber crops when compared with cereals-the next-most-important crop category in Nigeria. They showed that the national production of root and tuber crops was nearly four times that of cereals. Sanginga (2015) revealed that the aggregate value of yam, cassava, potato and sweet potato which are root and tuber crops exceeds all other staple crops, and is much higher than the value of cereal crops in Nigeria. There are many compelling reasons for encouraging root and tuber crops for sustainable food production in Nigeria. They are versatile staples to address food and nutrition security for millions of people, and produce more food per unit area of land (IITA, 2012) . Therefore, the cultivation of root and tuber crops is critical to food supply and food security in Nigeria.
Problem Statement The importance of tuber crops, especially cassava and yam, in ameliorating food insecurity has been highlighted by Food and Agriculture Organization (CGIAR, 1997) . Cassava and yam are capable in efficiently converting natural resources into a more usable product, caloric energy which is the highest of all major arable crops; almost double that of wheat and rice. Cassava and yam contribute to a more stable food system and predictable source of income (IITA, 2012) . However, tuber crops like cassava and yams are experiencing declining productivity trend (IITA, 2012; FAOSTAT, 2019) . The declining yield of cassava and yam needs to be addressed urgently because of their importance in reducing food insecurity. According to Mignouna et al (2015) efforts to boost cassava and yam yields are likely to be more sustainable and have a greater impact on food security than other crops. Agricultural credit is one of the vehicles to improve agricultural productivity in Nigeria (Onuselogu, 2014) . This is because, it will not only relax the capital constraints on the farm, it may also increase the ability of the farmers to adopt agricultural technologies and mechanization that can improve agricultural yields (Awotide et al, 2015) . Therefore, the study is aimed at analyzing the effects of agricultural credit on cassava yield in Nigeria. Specifically, the study estimated the long and short run effects of agricultural credit on cassava output and yield in Nigeria.
Study Area and Data Collection This area of study is Nigeria. Nigeria is located on the Gulf of Guinea in West Africa. Nigeria occupies an area of 923, 789 square kilometers and is bordered on the East by Republic of Cameroon, on the West by the Republic of Benin and on the North by Niger Republic. More than 70% of the population who reside in rural areas depend on agriculture as a means of livelihood (FAO, 2022) . Yam and Cassava are grown all over the country but mostly grown in Benue (known as the food basket of the nation), other state includes; Cross River, Adamawa, Delta, Ekiti, Imo, Edo, Kaduna, Ogun, Kwara, Ondo, Osun, Plateau and Oyo (Adetiloye, 2012) . Data for this study were collected mainly from secondary sources. Agricultural credit (million Naira), average rainfall (millimeters) data were extracted from Central Bank of Nigeria (CBN) Statistical Bulletin. Cassava Output and yield, Farm labour (modelled as the ratio of farmers' population to rural population in Nigeria), farmers population, rural population, fertilizer, arable land per farmer and temperature were extracted from FAOSTAT website (FAOSTAT, 2023) . All monetary values were deflated (GDP deflator) (obtainable from CBN Statistical Bulletin) using 2010 constant prices to exclude the influence of inflation, other temporary monetary and fiscal trends. The data range from 1981 to 2021. Summary Statistics of all the variables used in the study are presented in Appendix 1.
Data Analysis Techniques Descriptive statistics were applied on the relevant data collected for this study. The descriptive statistics applied include mean estimation and growth trend analysis. The growth rates of cassava output and yield were estimated following the procedure of Barrett (2001) . For example, the growth rate of cassava output was estimated using equation 1 as: log (Cassava t ) = o + ψ 1 (Year) + ---(1) Where log is the logarithm in base 10, Year is the period under consideration, where 1981 stands for 1 and 1982 stands for 2 and 41 represents 2021. The Cassava t is cassava output in each of the corresponding year, the error term is , ψ 1 is the estimated cassava output growth rate when expressed in percentage. Autoregressive distributed lag (ARDL) methodology was applied to relevant data collected for this study in measuring the long and short run effects of Agricultural credit on cassava production and yield. The first step in applying ARDL is the bounds test for the null hypothesis of no cointegration. Following the step of Udoh ( 2011 ), the null hypothesis of cointegration among the dependent variable and 2 ) assumes that Cassava yield (CAS) is determined by Rainfall (RAF), Temperature(TEM), land(LAN), farm labour (LAB), agricultural credit(CRE) and fertilizer (FER). Where p and q are the lag lengths of dependent and independent variables respectively. The long run elasticities are the c s , and a s are the short run elasticities. ∆ is the differencing factor and is the error term, log is logarithm in base 10. The null and alternative hypotheses for equation 2 can be stated as: H 0 : c 1 = c 2 = c 3 = c 4 = c 5 = c 6 = c 7 = 0 (no long run relationship among the variables) H 1 : c 1 ≠ c 2 ≠ c 3 ≠ c 4 ≠ c 5 ≠ c 6 ≠ c 7 ≠0 (there is long run relationship among the variables) After estimating equation 2, the bounds testing was done by testing H 0 against H 1 , the calculated F and tstatistics were then assessed against the critical values and approximate probability values given by Kripfganz and Schneider (2018) for small sample size estimation. The lower bound critical values assume that the explanatory variables are integrated of order zero, that is 1(0), while the upper critical values assumed that the explanatory variables are integrated of order one, that is 1(1). If the calculated F and T values are lower than the lower bounds, the null hypothesis is accepted. If they are greater than the lower bounds but less than the upper bounds, the decision is inconclusive. Finally, if the F and T values are greater than the upper bounds, the null hypothesis of no cointegration is rejected in favour of existence of a long-run relationship among the variables. Once the existence of a long run cointegration relationship is confirmed, the conditional ARDL (p1, q1, q2, q3, q4, q5, q6) long run equation for cassava yield model can be estimated as: + + + + + + ∑ +∑ ∑ ∑ +∑ + ∑ +∑ + --- (2) Equations ( = + + + + + + + - -- (3) The short run dynamic estimates can be obtained by estimating an error correction model associated with the long run parameters from equation 4 as: = +∑ ∑ ∑ ∑ +∑ + ∑ +∑ + ---(4) Where ECT is the error correction term, is the speed of adjustment to equilibrium, and are the long and short run elasticities that measure the effect of agricultural credit and other explanatory variables on cassava yield. Other variables are as previously defined. In pressing further with ARDL analysis, we performed Augmented Dickey -Fuller test (ADF) test to check for unit roots in order to be sure that none of the variables are in the integration order greater than one. Augmented Dickey-Fuller test shows that all the variables of interest were stationary after they were differenced, except temperature, labour and rainfall variables, which are stationary at levels. This means that all the variables are integrated of order one I(1), while temperature, labour and rainfall variables are of I(0). This is reported in Appendix 2. The relevant optimal lag lengths were also determined by Schwarz Information Criteria (SBIC) indicated in Appendix 3.
Result and Discussion of Cassava Production and Yield Trend Table 1 reveals that cassava output has increased from minimum of 10 million tonnes to 65 million tonnes between 1981 and 2021. The fact remains that the increase in cassava output is due to expansion of land put under cassava cultivation as the land under cassava cultivation increased from the minimum of 1060 hectares to 9780 hectares during the period under consideration. This became clearer as the table shows that cassava out and land expansion grew almost at the same rate (cassava and land cultivation under cassava grew at 4.50% and 5.08% respectively). This may explain why the cassava yield growth rate stood at -0.58%. FAO (2018) has indicated that increase in agricultural output in Nigeria comes mainly from increase in land expansion rather than through productivity gain. The table also indicates that the mean agricultural credit is 1.01 billion Naira which ranges from 0.23 to 4.60 billion Naira. It grew by about 23% within the years under consideration. Whether this positive growth in agricultural credit will have impact on cassava output or yield or not depends on how much this credit goes into cassava production activities. 2 shows that roots and tubers crop category in which cassava has the largest share in term of the amount and value has been given share of the of Agricultural Guarantee Credit Scheme Fund (AGCSF) that is less than their contribution to food crop sector. The tables reveals that grains received almost four time (357%) of its contribution to food crop sector, root and tuber received less than hundred percent(99%) of their contribution to food crop sector. If cassava production is not accorded the right priority in credit allocation, it may limit the impact of the credit on cassava output and yield. Other authors have also reported the negligence of roots and tubers in terms of financing and development in Nigeria (IITA, 2012; Sanginga, 2015) . Another factor that may affect the impact of agricultural credit on cassava production is the amount credit received per farmers. The lower the amount the lower may be the expected impact on cassava output and yield. Table 3 demonstrates that per capita agricultural credit has increased on the average from N5754 to N158914, the real value of per capita credit has declined from N15241 in 1981 to N1056.The implication of that is that farmers could only buy about 7% of what they were able to buy in 1981 in 2021(N1056/ N15241) with the given credit. The study has also indicates that per capita credit for farmers has declined from 1937 USD in 1981 to 735 USD in 2021 as presented in Table 3 . For farmers that depend on imported farms equipment and agrochemical, this also means they could only buy 38% (735/1937) in 2021 of farms equipment and agrochemical they purchased in 1991 with the agricultural credit they secured. This may partly explain the reason for low level of agricultural mechanisation in Nigeria. For example, while other developing countries have 130 tractors/100km 2 , Nigeria has seven (7) tractors/100km 2 compared with Sub Sahara Africa (SSA) and World averages of 13 tractors/100km 2 and 200 tractors/100km 2 of arable land respectively (WDI, 2019) . This implies that Nigerian farmers have low accessibility to means of agricultural mechanization that can increase their productivity. Oyakhilomen and Rekwot (2014) have also alluded to the negative effect of inflation on agricultural sector, because it reduces the purchasing power of the farmers. They recommended that the Central Bank of Nigeria should pay more attention to the trend of inflation and pursue policies that will ensure single digit inflation. The result of the estimation of the long and short run effects of agricultural credit on cassava output is presented in Table 3 . The maximum length selection was done based on SBIC and presented in Appendix 3. SBIC supports selection of maximum lag length of one (1) year. The optimal lag length structure selected for each of the variable and is reported in Appendix 4. The appendix 4 suggests that the optimal lag length structure is 1,1,0,1,1,0,1. This implies that cassava output, rainfall, temperature and labour will be lagged one (1) year, while the current forms of other variables such as agricultural credit, arable land and fertilizer will be used in the estimation of the model. Table 4 reveals that the estimated F value of 260.76 is significant at 1% level of significance. This is an attestation to the fact that the cassava output and agricultural credit model estimated in Table 3 is a good fit and therefore the model can be interpreted to represent the economic relationship among the variables specified in the table. The estimated adjusted R 2 of 0.9856 suggests that the explanatory variables specified in Table 3 can explain 99% variation in cassava output. Table 4 reveals that the significant determinants of cassava output on the long run are past cassava output, annual rainfall, past and current arable land, past and current labour and fertilizer. The importance of past output in stimulating current output has been associated with income opportunity generated in the past production, which can be reinvested in the current production activity. The volume of the past cassava output can be an indication of past farmers' experiences which can enable the farmers to avoid the negative past agricultural production decision and adopt the best farming practices. The coefficient of lagged rainfall is significant but negative because excessive rainfall can be detrimental to cassava production. The negative effect of last year (2022) flood in Nigeria was a clear evidence of the detrimental effect of excessive rainfall on agricultural production (Reed et al, 2022) . Flood can hinder some farmers from planting and harvesting their crops. Land is the factory where agricultural production takes place, hence the positive and significant coefficient of arable land in Table 3 . In the same token, a degraded land can be less productive. This may explain the negative coefficient of the lagged land because it will take current serious efforts to improve unfertile and degraded land. Philip et al (2019) has also reported unproductivity of Nigerian land because of shortened fallow period and little investment on the farm to restore land fertility. Table 4 also reports a positive and significant coefficient of current labour variable in the model. This is because agriculture activity cannot take place in forms of cultivation of land, planting and harvesting without labour. However, labour may be less productive if the farmers are sickly and too old to farm productively. This may be account for the negative coefficient of the lagged labour variable in the model. Other scholars have reported unproductivity of farm labour in Nigeria (Wisdom, 2016; Angaye, 2016) . If the low agricultural labour productivity is not addressed it can lead to labour migration from rural area to urban area and diversification from agricultural sector to non-farming enterprises (Sackey, et al, 2012; Takeshima, et al, 2018) . Fertilizer coefficient is positive and significant as expressed in Table 4 . This is because fertilizer is important in increasing land productivity (Jaja and Barber, 2017) . The table also shows that agricultural credit is positively related with cassava output but not significant even at 10% level of significance. This may be due to low-level of agricultural credit flow to agriculture from ACGSF (Olowofeso et al, 2017) . Nwakanma et al (2014) has also indicated a positive but non-significant long-run relationship between agricultural output and credit in Nigeria. Evidence suggests that the financing gap for smallholder farmers in Nigeria is huge (AfDB, 2016) . The reason for non-significance of agricultural credit can be attributed to the fact that majority of the farmers are financially excluded (Alabi et al, 2020) . Evidence shows that majority of the farmers relied on informal credit sources with smaller amount of loans that is too little to significantly increase their production (Awotide et al, 2015) . Awotide et al (2015) reported that agricultural credit may not affect agricultural production and productivity directly but the indirect effect may reflect on the ability of agricultural credit to influence adoption of farm investment that may increase farm productivity. However, we put agricultural credit in the estimated equation to check if agricultural credit will behave differently from the hypothesis of Awotide et al (2015) . In Table 4 , the error correction term of -0.3037 is significant at 1% level of significance. This confirms a joint significant relationship among the variables estimated in Table 3 . The fact that the value is -0.3037 implies that about 30% of dis-equilibrium to the long run relationship in the model can be reverted within a year. In addition, the short run model of cassava output and agricultural credit presented in Table 4 reveals that arable land, labour and fertilizer have short significant effects on cassava output in Nigeria. In estimating the effect of agricultural credit on cassava yield in Table 5 , maximum lag length for the cassava yield and agricultural credit model was determined and presented in Appendix 5. SBIC criterion supports maximum lag selection of one (1) as presented in Appendix 5. The optimal lag length structure for each of the variable in the model was also determined. Appendix 4 shows that the optimal lag length structure for the model is 1,0,1,0,0,0,0. Suggesting that cassava yield and arable land should be lagged one (1) year, while the current values of rainfall, temperature, labour, agricultural credit and fertilizer were used in the estimation. The cointegration of relationship among the variables was tested using Bound test and presented in Appendix 6. Since the calculated F and T values of 3.833 and -4.563 respectively are greater than the theoretical value of F and T of 3.665 and -4.017 (in absolute terms) respectively, the alternative hypothesis of cointegration among the variables is accepted. This means that long and short runs relationship among the variables can be estimated. Table 5 presents ECT of -0.7085, which is significance at 1% level of significance. The significance of ECT is a confirmation of joint significance of the variables in the model. Table 5 reveals that the estimated F value of 7.22 is significant at 1% level of significance. This is an evidence to show that the model estimated in Table 5 is a good fit and can be used to represent the economic relationship between cassava yield, agricultural credit and other explanatory variables such, past cassava yield, rainfall, temperature, arable land, labour and fertilizer. The adjusted R 2 of 0.5670 indicates that about 57% of variation in cassava yield can be explained by the explanatory variables stated in Table 5 . The table reveals that past cassava yield has positive and significant effect on the current cassava yield. The positive effect of past agricultural activities on the current activities has been explained to be due to the backward integration of income received from the past agricultural production into the current production. Therefore, as expected the past cassava yield has positive and significant coefficient on the current cassava yield. As in cassava production, excessive rainfall can affect cassava yield as cassava need moderate rainfall for optimal performance. This may explain the negative rainfall coefficient as it relates to cassava yield presented in Table 5 . The table also shows that past temperature is a positive and significant determinant of cassava yield. This is expected as cassava needs adequate temperature with associated sunshine for improved performance. Akinwumiju, et al (2020) have indicated that the highest production cassava is expected in the tropics, with temperature and annual rainfall amount ranges of 25-27 C and 1200-1500 mm, respectively. They have proved through greenhouse and on-farm experiments that cassava has a wider tolerance range for temperature (15-35 C) and rainfall (500-5000 mm). The coefficient of labour is significant but negative. This is an indication of less productivity of labour. Other authors that have reported unproductivity of farm labour in Nigeria include Wisdom (2016) and Angaye (2016) . The unproductivity of agricultural labour has been adduced for urban-rural migration and disinvestment in agricultural sector (Sackey, et al, 2012; Takeshima, et al, 2018) . The table also reports a non-significance of agricultural credit because the farmers are disadvantaged in accessing agricultural credit because of the nature of farming and their locations that may lead to their financial exclusion. Table 5 also demonstrates that on the short run, rainfall land and labour have significant but negative relationship with cassava yield. It has been explained that excessive rainfall may have detrimental effect on cassava output and yield. The negative but significant coefficients of land and labour have been explained on the account of declined land and labour productivities being witnessed in agricultural sector in Nigeria.
CONCLUSION AND RECOMMENDATIONS It is evident from the study that agricultural credit does not have significant long and short run effects on cassava production and yield. The reason for the non-significance of agricultural credit has been attributed to many factors in the study. In addition, the study submitted that fertilizer is a positive determinant of cassava production on the long and short run but not on the cassava yield. The declined in real value of per capita credit can be addressed by increasing the amount of credit given to the farmers. There is also the need to design more credit facilities suitable farmers. The current Anchor Borrowers programme should be designed to capture more resource poor cassava farmers. The farmers should be encouraged to reinvest the profits they made from cassava farming back into cassava production. This will help farmers reap the full benefits of their investment in cassava production.
Table 1 : Growth in Cassava Output, Cassava Yield and Agricultural Credit in Nigeria 1 Variable Mean Standard Minimum Maximum Growth Rate (%) Deviation Cassava Output (Million tonnes) 35.60 16.50 10.00 65.00 4.50 Cassava Yield (Tonnes/Ha) 9.99 1.56 5.82 12.22 -0.58 Cassava Land Area(Hectares) 3817.32 2331.12 1060.00 9780.00 5.08 Credit (Million Naira) 1011.82 1332.58 0.23 4600.00 23.08 Source: Computed by the Authors Table
Table 2 : Share of Root and Tubers in Food Crops Sector and ACGSF Credit (1981-2021) 2 Food Crops Share in ACGSF Share in Food Crops Agriculture Credit Sector Orientation Index(AOI) Grains 50.88 14.24 3.57 Roots and Tubers 48.34 48.80 0.99 Beans and Soya 0.25 1.67 0.15 Vegetables 0.54 6.82 0.08 Source: Computed from CBN (2021) and FAOSTAT (2023)
Table 3 : Average Per capita Agricultural Credit in Naira and US Dollars (USD) (1981-2021) 3 Period Amount Per capita (Naira) Real Per Capita (Naira) Per capita (USD) ('000) 1981-1990 68959 5754 15241 1937 1991-2000 181048 10488 828 373 2001-2010 4533025 101713 1550 767 2011-2021 7654015 158914 1056 735 3220109 73509 4581 465 Source: Computed by the Authors
, ADEGBOYEGA EYITAYO OGUNTADE AND REUBEN ADEOLU ALABI The Effect of Agricultural Credit on Cassava Output and Yield
Table 4 : Estimates of the long and short run coefficients of the ARDL model (Effect of Agricultural Credit on Cassava Output) 4 , () and (*) expresses 10%, 5% and 1% significance level respectively, ∆ = differencing factor. (-1) represents lag length of one (1) year Source: Computed by the Authors Long Run Short Run F 260.76 Prob > F 0.0000 R 2 0.9894 R 2 0.6216 Adj R 2 0.9856 Adj R 2 0.4865 Variables Coefficient P>|t| Variables Coefficient P>|t| Cassava Output(-1) 0.6963* 0.000 ∆Rainfall -0.1280 0.395 Rainfall -0.1280 0.395 ∆Temperature -0.0010 0.980 Rainfall(-1) -0.3377 0.025 ∆Arable Land 18.9933* 0.002 Temperature -0.0010 0.980 ∆Labour 17.9221* 0.003 Arable Land 18.9932* 0.002 ∆Agric credit -0.0026 0.889 Arable Land(-1) -17.9031 0.002 ∆Fertilizer 0.1022* 0.002 Labour 17.9221* 0.003 ECT -0.3037* 0.009 Labour(-1) -17.3187* 0.003 Agric Credit -0.0026 0.889 Fertilizer 0.1022* 0.002 Constant -14.6100 0.239 Log likelihood 58.1923 (*)
Table 5 : Estimates of the long and short run coefficients of the ARDL model (Effect of Agricultural Credit on Cassava Yield) 5 , () and (*) expresses 10%, 5% and 1% significance level respectively, ∆ = differencing factor. Source: Computed by the Authors Long Run Short Run F 7.22 Prob > F 0.0000 Prob > F 0.0000 R 2 0.6581 R 2 0.4726 Adj R 2 0.5670 Adj R 2 0.3320 Variables Coefficient P>|t| Variables Coefficient P>|t| Cassava Yield(-1) 0.2915* 0.070 ∆Rainfall -0.4219* 0.079 Rainfall -0.4219* 0.079 ∆Temperature 0.0578 0.366 Temperature 0.0578 0.366 ∆Arable Land -0..9930 0.025 Temperature(-1) 0.1269 0.046 ∆Labour -1.3335* 0.012 Arable Land -0.9931 0.025 ∆Agric Credit 0.0305 0.294 Labour -1.3335* 0.012 ∆Fertilizer -0.0410 0.413 Agric Credit 0.0305 0.294 Constant 42.8945* 0.003 Fertilizer -0.04103 0.413 ECT -0.7085* 0.000 Constant 42.8945* 0.003 Log likelihood 36.3203 (*)
|
10.1038/s41598-019-49590-3
|
cc-by
| null | null |
openalex
|
Arabidopsis VIRE2-inteRActinG pROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsis vip2 mutants are recalcitrant to Agrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly, AtVIP2 overexpressor lines in Arabidopsis did not show an improvement in Agrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. in addition to regulating genes involved in Agrobacterium-mediated plant transformation, AtVIP2 overexpressor line showed differential expression of genes involved in abiotic stresses. the majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated in AtVIP2 overexpressor lines. Consistent with this observation, AtVIP2 overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role in Agrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.The plant pathogen Agrobacterium tumefaciens causes neoplastic growth called crown galls on plants by transferring genetic material coded on the transfer DNA (T-DNA) from the tumor-inducing (Ti plasmid) to plant cells, resulting in the genome modification following integration (see reviews ). The genetic transformation of a plant cell by A. tumefaciens involves the synthesis and translocation of the T-DNA mediated by the virulence (vir) gene products, interaction of the translocated virulence proteins with their cognate partners in the host, and expression of the genes on the T-DNA following integration (see reviews 1, 2, 5, 6 ). Some of the virulence proteins, namely VirD2, VirD5, VirE2, VirE3, and VirF, are A. tumefaciens effector proteins that are directly translocated into the plant cell via the type IV secretion system (T4SS) 7, 8 . One of the translocated virulence proteins, VirE2, binds to the single-stranded DNA (ssDNA), in vitro, to form a telephone-cord like structure protecting it from degradation by nucleases . Plant proteins that interact with VirE2 were surveyed by the yeast two-hybrid system, resulting in the identification of two VirE2-interacting proteins (VIP), VIP1 and VIP2 13 . VirE2 is suggested to piggy-back on VIP1 for nuclear import via an importin α-dependent pathway and this process is up-regulated by a host MAP kinase that phosphorylates VIP1 14 . VIP2 is a negative on TATA-less (NOT)-domain containing protein which likely functions as a transcription factor and is required for plant stable transformation, but not for transient T-DNA expression 15 . An Arabidopsis vip2 mutant was shown to be deficient in T-DNA integration due to changes in the expression of many genes, including core histones, suggesting that VIP2 has a role in T-DNA integration 15 . A NOT2 homolog in yeast acts as a general negative regulator of gene expression. Similarly, in Drosophila, a NOT2 homolog, the Rga protein, has probable function in mediating interaction between chromatin proteins and the transcriptional complex 16, 17 . NOT2 is the core member of the CCR4-NOT complex that regulates mRNA metabolism at both transcriptional and posttranscriptional levels 18 and has a role in promoting transcriptional elongation by RNA polymerase II 19, 20 . The NOT proteins (VIP2/NOT2b and NOT2a) are general transcriptional regulators essential for plant development. NOT2s acts as a scaffold to interact with RNA polymerase II, and promotes transcription of both protein coding and miRNA genes, and facilitates efficient DICER-LIKE1 recruitment in miRNA biogenesis 21 . VIP2/NOT2b interact with miRNA processing factors such as cap binding proteins, CBP80 and CBP20 21 , and the interaction is modulated by VirD5 22 . Here, we demonstrate that VIP2 is a transcription regulator and overexpression of VIP2 in Arabidopsis modulates expression of genes not only involved in Agrobacterium-mediated plant transformation but also genes associated with several other pathways including abiotic stresses. This finding is consistent with its role as transcription regulator. Interestingly, overexpression of VIP2 in Arabidopsis did not significantly increase Agrobacterium-mediated root transformation efficiency.
Results VIP2 is a transcriptional regulator. Based on the VIP2 sequence, transcript profiling data of Arabidopsis vip2 mutant 15 , and nuclear localization data, we hypothesized that VIP2 is a transcriptional regulator. To explore this hypothesis, we first tested whether VIP2 can activate transcription in yeast when bound to DNA as previously described for VirE3 23 . We therefore tested the VIP2 protein for transcriptional activation through yeast one-hybrid assay 24 . For this purpose, the VIP2 (GenBank # AF295433) open reading frame was fused to the GAL4 DNA binding domain (DBD) and cloned into a pGBKT7 yeast vector. The human lamin C (LamC) and topoisomerase I (TopI) genes cloned into the pGBKT7 vector were used as controls. We performed single and double dropout assays to confirm the DNA binding ability of the VIP2 protein in yeast. Undiluted and a 1:10 dilution of all the four clones were able to grow well on solid synthetic dropout (SD) media lacking tryptophan. However, 1:100 and 1:1000 dilutions showed little to no growth (Fig. 1a ). The VIP2 fusion construct grew on media lacking Trp and His and containing 10 mM 3-amino-1,2,4-triazole (3-AT), while the LamC and TopI did not (Fig. 1a ). These results suggest that the VIP2 protein can activate and express the his3 gene, allowing yeast to grow on a histidine and tryptophan-deficient medium. The above findings suggest VIP2 can activate transcription in yeast, and therefore potentially is a transcription regulator. To validate the transcription activation property of VIP2 in planta, a promoter trans-activation assay, as described earlier 25 , was used. Tobacco leaves were bombarded with different combinations of reporter constructs such as pSAT6-uasP-GUS only, pSAT6-uasP-GUS and pSAT6-mGAL4-DBD constructs and, pSAT6-uasP-GUS and pSAT6-mGAL4-DBD-VIP2 constructs (Fig. 1b ). GUS histochemical staining was performed 48-72 h post DNA bombardment, and the following observations were recorded. We observed few blue spots in the controls, pSAT6-uasP-GUS alone (32 ± 8 blue spots) or pSAT6-uasP-GUS vector expressing the unfused GAL4 DBD (pSAT6-mGAL4-DBD; 69 ± 12 blue spots) and weak expression of the GUS reporter (Fig. 1b ). In contrast, co-bombardment of pSAT6-uasP-GUS with pSAT6-mGAL4-DBD-VIP2 that expresses mGAL4-VIP2 fusion from the CaMV35S promoter resulted in a significantly higher number of blue spots (685 ± 181 blue spots) and increased expression of the GUS reporter (Fig. 1b ). Based on the above results we hypothesized that VIP2 is efficient in transactivation of the UAS promoter when fused to a DBD. These results further confirmed that VIP2 functions as a transcriptional regulator in planta, corroborating the findings obtained in the yeast-one hybrid assay (see Fig. 1a ).
VirE2 interacts with the C-terminal NOT domain of VIP2 in yeast expression system. VIP2 was identified as a VIRE2 interacting protein using a yeast two hybrid system 13, 26 . However, it was not clear which domain of VIP2 interacts with VirE2. It is especially important to know if the NOT domain is important for interaction. To determine this, the VIP2 gene was split into two regions; a C-terminal fragment containing the NOT2/NOT3/NOT5 plus 445 bp at the 3′ end and an N-terminal fragment minus the NOT domains (5′ end 1440 bp) as described 15 . Based on yeast growth, we concluded that VirE2 interacts with 3′ end of VIP2 that contain the NOT domain (Fig. 2a ). In addition, both VIP2 and VirE2 proteins can form a dimer with themselves and activate the reporter genes (Fig. 2a ). LacZ expression was quantified by β-galactosidase activity to further confirm protein-protein interactions (Fig. 2b ). Rescuing the tumorigenesis-deficiency phenotype in the Atvip2 mutant with constitutive expression of the AtVIP2 gene. Previously, it was reported that the Atvip2 mutant is recalcitrant to stable transformation. However, complementation of the mutant was not shown 15 . Therefore, it is not definite if the transformation recalcitrance phenotype is due to the loss of function of VIP2. To address this, a construct containing the AtVIP2 gene (accession # AT5G59710.1) driven by the CaMV35S promoter was transformed into the Atvip2 mutant line. T1 plants of three independent Atvip2 lines expressing the AtVIP2 gene (Atvip2::35S-2, Atvip2::35S-3 and Atvip2::35S-5) were tested for restoration of the tumorigenesis-susceptibility phenotype. Root segments of all three transgenic lines along with the wild-type and the Atvip2 mutant were inoculated with a tumorigenic A. tumefaciens strain as described 15 . Four weeks after Agrobacterium inoculation, number of tumors produced in each plate was scored. As expected, the wild-type Col-0 was susceptible to transformation and Atvip2 was recalcitrant. The three transgenic Atvip2 lines expressing the AtVIP2 gene produced tumors at similar frequencies to that of Col-0 (Fig. 3a ), indicating complementation of the tumorigenesis-deficiency phenotype of the Atvip2 mutant. Furthermore, another stable transformation assay with a non-tumorigenic A. tumefaciens strain with uidA gene within its T-DNA was performed on Atvip2 lines expressing the AtVIP2 gene as previously described 27, 28 . GUS staining of calli indicated increased expression of the uidA gene in the complemented line compared to the Atvip2 mutant and the uidA expression was comparable to that of Col-0 (Fig. 3b ). The data from this stable transformation assay are in accordance with the tumorigenesis data and further strengthen our conclusion that expression of the AtVIP2 gene in the Atvip2 mutant could restore the transformation-susceptibility phenotype. Taken together, these data suggest that mutation in the AtVIP2 gene in the Atvip2 mutant is responsible for the transformation recalcitrant phenotype.
VIP2 is ubiquitously expressed in major plant organs in arabidopsis. Expression of the YFP-tagged AtVIP2 gene in Arabidopsis indicated the cell and tissue-specific expression of the AtVIP2 gene in the female gametophyte (megasporocytes and tapetum cells) organs, and not in other plant organs 29 . RT-PCR in the same samples showed expression of the AtVIP2 gene in mature flowers and flower buds, not in leaves, stem and roots 29 . Interestingly, we detected the expression of the AtVIP2 gene by semi-quantitative RT-PCR in many plant organs including young roots, leaves, stem, petioles and floral tissues (Fig. 4a ). These findings contradict previously suggested expression of the AtVIP2 gene in specific Arabidopsis organelles 29 . To further confirm the AtVIP2 expression, Arabidopsis transgenic plants expressing an AtVIP2 Promoter:uidA fusion (Fig. 4b ) were developed and 3-4 independent transgenic plants were analyzed by histochemical GUS staining (Fig. 4c ). We observed the expression of the AtVIP2 promoter to be ubiquitous in most plant organs and specifically in the following tissues-: roots, root hairs, leaves, shoots, trichomes, and floral organs (Fig. 4c ). Further, based on the GUS staining we concluded that the expression of AtVIP2 is significantly higher in vasculature and root tissues including lateral roots and root hairs. Additionally, we compared the expression of the AtVIP2 promoter with the CaMV35S promoter. Interestingly, GUS staining in AtVIP2 Promoter:uidA expressing plants was much stronger in many plant organs when compared to the transgenic Arabidopsis plants expressing a CaMV35S-uidA construct (Supplementary Fig. S1 ). Taken together, these data illustrate that the AtVIP2 gene is ubiquitously expressed in most plant cells and tissues. Our observations are consistent with the information provided at https://apps.araport.org/thalemine/portal.do?externalids=AT5G59710 (Supplementary Fig. S2 ). More interestingly, we report here the discovery of a plant specific promoter that is potentially stronger and ubiquitous in expression when compared to the CaMV35S promoter. transgenic Arabidopsis plants overexpressing the AtVIP2 gene did not show enhanced transformation. Since the Atvip2 mutant is recalcitrant to Agrobacterium-mediated root transformation, we were interested to determine the effect of overexpressing AtVIP2 on root transformation. Transgenic Col-0 lines overexpressing AtVIP2 driven by the CaMV35S promoter were generated (Supplementary Fig. S3 ). A quantitative root tumor assay, as described above, was done using the tumorigenic A. tumefaciens strain A208. Tumor formation was monitored for a period of four weeks and data were recorded. Our results suggested that overexpression of AtVIP2 did not significantly improve transformation (Supplementary Figs. S4a, upper panel and S4b, left). The effect of AtVIP2 overexpression on another stable transformation assay was determined by calculating the frequency of PPT-resistant calli following infection with a disarmed A. tumefaciens strain GV3101 (pCAS1) as previously described 28, 30 . There was no significant difference observed in the frequency of PPT-resistant calli formation between AtVIP2 overexpressing transgenic lines and vector control/or Col-0 wild-type (Supplementary Figs. S4a, lower panel and S4b, right). Taken together, these data suggest that overexpression of AtVIP2 in Arabidopsis does not improve root transformation.
VIP2 regulates transcription of defense genes, histones and galactolipid biosynthetic genes. To investigate the role of VIP2 in Agrobacterium-plant interaction, we carried out transcriptome profiling by comparing a homozygous Arabidopsis AtVIP2 overexpressor line with wild-type Col-0 plants following Agrobacterium infection. Soil grown Arabidopsis plants in the vegetative stage with fully formed rosette leaves were syringe infiltrated with the disarmed strain of A. tumefaciens, GV3101 harboring the uidA-intron gene within its T-DNA (OD 600 = 0.2 15 ), and samples were collected at 0, 48 and 72 h after infection (HAI). RNA extracted from these samples were analyzed for gene expression using whole genome Affymetrix gene chip (ATH1) microarray. A total of 1,634 genes were differentially expressed (DE) between AtVIP2 overexpressor line and Col-0. To validate the microarray data, expression of 10 DE genes from AtVIP2 overexpressor plants were analyzed by RT-qPCR (Supplementary Fig. S5 ). The results obtained from RT-qPCR are in general agreement with the microarray results. The Venn diagram for DE genes showed that many DE genes during Agrobacterium infection overlapped with DE genes of AtVIP2 overexpressor plants without Agrobacterium infection (Fig. 5a ). To study the functional significance of all DE genes, we classified them based on Gene Ontology (GO) term enrichment (Fig. 5b ). Various functional categories such as protein targeting to membrane, circadian rhythm, signaling, response to stimulus and regulation of hypersensitive response were enriched. Interestingly, GO terms associated with molecular functions such as sequence-specific DNA binding transcription factor activity and transcription regulatory region DNA binding were enriched. This shows the involvement of VIP2 in transcriptional regulation upon Agrobacteriuminfection with a T-DNA transfer competent strain. Many GO terms associated with stress or immune response were also observed. Interestingly, the majority of the DE genes in AtVIP2 overexpressor were from 0 HAI i.e., without Agrobacterium infection. Surprisingly, many of these genes were also DE in wild-type Col-0 upon Agrobacterium infection (Supplementary Fig. S6 , Supplementary Table S1 ). GO term analysis of these commonly DE genes showed that they belonged to various functional categories such as ethylene-mediated signaling, response to ethylene, response to water, cellular response to hormone and defense response. Many genes in these GO terms are known to be involved in plant defense against various stress conditions 31 . Genes regulated by Agrobacterium infection and AtVIP2 overexpression include genes that encode WRKY70, receptor-like protein kinase THESEUS 1, Calmodulin-binding protein 60-like G (CBP60G), Calmodulin like 42 (CML42), Pathogenesis-related protein 5 (PR5), MATE efflux family protein, APS reductase 3, WRKY30 etc. These data suggest that at gene expression level, overexpression of AtVIP2 in Arabidopsis mimics gene regulation during Agrobacterium infection in wild-type Col-0. From the Mapman analysis, we additionally identified many genes involved in proteolysis that were down-regulated at 0 HAI in AtVIP2 overexpressor plants (Supplementary Fig. S7 ) compared to Col-0. The above data further strengthens the role for VIP2 as a transcriptional regulator. An elf-18 inducible gene, PP2-A5 (phloem protein 2 A5; At1g65390) 32 , was one the genes showing the highest level of induction in AtVIP2 overexpressor plants upon Agrobacterium infection. Some of the other genes induced are CAF1A (CCR4-associated factor 1a; At3g44260), β-glucosidase 18 (At1g52400), QQS (Qua-Quine Starch; At3g30720), and PCC1 (Pathogen and Circadian Controlled 1; At3g22231). Some of the genes with reduced Genes that were specifically regulated in AtVIP2 overexpressor plants during Agrobacterium infection process (AtVIP2 overexpressing plants at 48 and 72 HAI compared with AtVIP2 overexpressing plants at 0 HAI) belonged to GO terms such as galactolipid biosynthetic process, cellular response to phosphate starvation, response to fructose, response to sucrose and UDP-glycosyltransferase (UGT) activity (Supplementary Table S2 ). These GO categories were observed from the down-regulated data set. UGTs transfer glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones) containing an aromatic ring 33 . They were found to be involved in plant disease and defense responses. We also found that various histone genes that have been shown to be down-regulated in an Atvip2 mutant 15 and a not2a Atvip2 mutant 21 , are up-regulated in our data set (Supplementary Fig. S8 ). Some up-regulated genes identified in our study encode proteins such as Arabinogalactan, CAF1, Nodulin-Like Protein and Protein Phosphatase 2 C which have been previously reported to be important for Agrobacterium-mediated plant transformation . ABRe motif containing genes are repressed in AtVIP2 overexpressors. Cis-acting elements are key regulators of gene expression. Therefore, in silico motif analysis to identify motifs that are specifically enriched in the promoters of DE genes was performed. Enrichment of mCACGTGk motif in the down-regulated gene set was identified (Supplementary Tables S3, S4 ). ACGTG is a core of the Abscisic Acid Responsive Element (ABRE) that is involved in the abscisic acid (ABA)-regulated gene expression 38 , and genes containing them responds to abiotic stress including drought stress and ABA treatment 39 . ABA, an abiotic stress hormone, has a major role in regulation of physiological processes during abiotic stress responses 40 . Some reports suggest that ABA is also involved in plant defense signaling against pathogens and is an essential component in integrating and fine-tuning abiotic and biotic stress-response signaling networks 41, 42 . The above reports are in support of the GO term analysis, in that many of the signaling pathways enriched in our study (Fig. 5 ) have cross-talk with each other (reviewed in 43 ). On the other hand, we did not find significantly enriched motifs in up-regulated gene set. AtVIP2 overexpression causes low ABA content and sensitivity to abiotic stresses. Since we observed enrichment of ABRE motifs in the promoters of down-regulated genes of AtVIP2 overexpressor plants, we further determined the ABA levels and the response of the AtVIP2 overexpressor lines to abiotic stress. We quantified the level of ABA in wild-type Col-0, AtVIP2 overexpressor, and Atvip2 knockout plants grown on half strength MS for three weeks. AtVIP2 overexpressor lines showed significantly less ABA content than Col-0 plants (Fig. 6 ). In addition, we studied response of Atvip2 and AtVIP2 overexpressor plants to various abiotic stresses by transplanting seedlings to half strength MS plates containing 100 mM NaCl, 5 μM ABA, 10 μM ABA or 75 mM mannitol. AtVIP2 overexpressor plants had lower fresh weights compared to wild-type plants when grown in 5 μM ABA, 100 mM NaCl or 75 mM mannitol (Fig. 7 ). The above data suggest that AtVIP2 regulates abiotic stress responses in plants, which is consistent with the speculated role as transcription regulator.
Discussion Agrobacterium-mediated plant transformation is a complex process involving the functions of both bacterial virulence proteins and host proteins in various steps of the transformation process. Roles for plant genes in T-DNA transfer and integration during the transformation process have been shown or proposed . VIP2 is one such plant gene required for Agrobacterium-mediated stable plant transformation 15 and acts as a general transcriptional regulator controlling plant development 21 . In the present study, surprisingly, we showed that overexpression of AtVIP2 in Arabidopsis did not have a significant impact on Agrobacterium-mediated plant transformation. Our finding contradicts the data from Zhao and coworkers 47 that illustrates up to 2.5-fold increase in Agrobacterium-mediated transformation efficiency in tobacco by the heterologous expression of the Triticum aestivum VIP2. It is not clear if endogenous VIP2 is expressed at saturated levels in tobacco. Nevertheless, these results show that over-expression of VIP2 can enhance transformation in some plant species, further confirming the role of VIP2 in Agrobacterium-mediated plant transformation. Another possible reason for the differences observed between our study, and Zhao et al. 47 may be the use of homologous versus heterologous VIP2 gene sequences for overexpression. In a previous study, it has been shown that overexpression of AtVIP1 (a bZIP protein) in Arabidopsis did not alter either transient or stable transformation susceptibility 48 , whereas T-DNA transformation efficiency was improved when AtVIP1 expressing transgenic tobacco plants were retransformed with Agrobacterium 49 . Though the increased transformation efficiency was attributed to the lack of use of full-length VIP1 cDNA 48, 49 , it is interesting to note that homologous expression of VIP1 did not alter transformation efficiency in Arabidopsis similar to our study. Based on the above findings, we speculate VIP2 is necessary but not a rate-limiting protein in Agrobacterium-mediated transformation in Arabidopsis. A previous study 22 showed that VirD5 interacts with AtVIP2 and competes with it for binding with cap binding proteins (CBPs; CBP20 and CBP80) that negatively regulates the Agrobacterium infection process. The interaction between AtVIP2 and VirD5 is very specific in that VirD5 interacts with AtVIP2 of Arabidopsis, but not with the homolog NOT2a of Arabidopsis or the ortholog OsNOT2 of rice 22 . It would be interesting to understand the role of AtVIP2 and VirD5 interaction in Agrobacterium-mediated plant transformation. In our previous publication, we demonstrated that VIP2 interacts with VIRE2 15 . Wang et al. 22 also showed that VIP2 and VIRE2 along with VIRD5 form a ternary complex in vivo. In this study, we specifically showed that VIRE2 interacts with C-terminal NOT domain of VIP2. We speculate that VIRE2 interacts with VIP2 to alter VIP2's activity, which in turn will regulate expression of genes such as histones to enhance Agrobacterium-mediated plant transformation. In this study, we also investigated AtVIP2 promoter activity in multiple tissues or organs and showed that the AtVIP2 promoter is highly active and expressed constitutively. Agrobacterium infection was previously shown to induce AtVIP2 expression in Arabidopsis 15 . We speculate that the VIP2 protein is present at saturated levels in Arabidopsis and therefore overproduction of this protein does not have an impact on plant transformation. However, overexpression of AtVIP2 resulted in differential expression of many genes that are important for Agrobacterium-mediated plant transformation (Fig. 5 ) including up-regulation of histone genes (Supplementary Fig. S8 ). In addition, expression of defense related genes were modulated in the AtVIP2 overexpressor plants. During Agrobacterium infection process, plant genes necessary for the transformation process are induced and host defense genes are repressed 50, 51 . Surprisingly, we also found many genes involved in proteolysis were down-regulated in AtVIP2 overexpressor plants (Supplementary Fig. S7 ). Proteolysis of proteins coating the T-DNA is important for subsequent integration of T-DNA to the host genome 52 . It would be interesting to study how VIP2 affects proteolysis. Genome-wide transcript profiling of Agrobacteriuminfected AtVIP2 overexpressor lines revealed the presence of many differentially regulated genes and GO terms. UGT is one of the GO terms specifically enriched in AtVIP2 S2 ). Overexpression of barley UGT in wheat resulted in enhanced resistance to Fusarium graminearum 53 . Recently, it has been shown that expression of wheat UGT in tobacco and Arabidopsis reduced the efficiency of Agrobacterium-mediated plant transformation 54 . Though we did not find any relevant literature related to the galactolipid biosynthetic process or cellular response to phosphate starvation in relation to Agrobacterium-mediated plant transformation, it is important to note that alteration of membrane lipid composition is one of the adaptive mechanisms in higher plants to cope with phosphate starvation 55 . Highly induced genes in AtVIP2 overexpressor plants upon Agrobacterium infection include CAF1A, β-glucosidase 18, QQS and PCC1. CAF1A is involved in mRNA deadenylation and mediation of stress responses 56 . β-glucosidase 18 plays a role in the metabolism of abiotic stress hormone ABA 57 . An orphan gene, QQS, functions in modulation of carbon and nitrogen allocation 58 . RNAi silencing of PCC1, a regulator of defense against pathogens and stress-activated transition to flowering, resulted in plants being more susceptible to hemi-biotrophic oomycete pathogen, Phytophthora brassicae, and more resistant to the necrotrophic fungal pathogen Botrytis cinerea 59 . GO terms related to abiotic stress responses such as response to salt stress, response to ABA, etc. are enriched among DE genes (Fig. 5 ). Motif analysis of DE genes dataset resulted in identification of ABRE containing motif (mCACGTGk) in the promoters of the down-regulated genes (Supplementary Table S3 ), and enrichment of these motifs in the 72 HAI down-regulated DE genes dataset (Supplementary Table S4 ). At 72 HAI, many abiotic stress responsive genes such as late embryogenesis abundant proteins (LEA) (At5g06760, At3g15670, At1g52690 and At2g35300), and calmodulin-like protein 41 (CML41) (At3g50770) were down-regulated. Endogenous ABA levels are an important factor in shaping plants tolerance to various abiotic stresses . ABA induces expression of ABA-responsive genes via ABREs in their promoter regions 60, 63 . ABRE containing elements are enriched in promoters of genes that are responsive to various abiotic stresses 64 . LEA proteins are intrinsically disordered proteins, have major role in abiotic stress tolerance in plants 65, 66 . CMLs are major Ca 2+ sensors, and they target many genes involved in various developmental processes and stress tolerance 67, 68 . CML41 was induced by both plant cell wall derived oligosaccharides and bacterial flagellin peptide Flg22, and have been suggested to have a role in dampening plant immune responses 69 . In addition, recently, CML41 has been shown to have increased expression in response to elevated temperature 70 . Expression of TSA1 and DEFL was also reduced in AtVIP2 overexpressor plants upon Agrobacterium infection. TSA1 is involved in MeJA-induced ER body formation in plants 71 . Similar to our study, infection of a fungal pathogen Alternaria brassicicola represses the expression of DEFL 72 . Results from our study along with previous reports 15, 21 showed that VIP2 is not only a regulator of plant genes involved in Agrobacterium-mediated plant transformation process but also a general transcriptional regulator that control multiple pathways. Our results showed that overexpression of AtVIP2 in Arabidopsis resulted in differential regulation of many abiotic stress related genes. This observation is consistent with the observation that the NOT2 domain of Fusarium oxysporum is suggested to regulate vegetative growth, conidiogenesis and virulence of the fungus by the transcriptional regulation of genes involved in multiple pathways controlling cell wall integrity, oxidative stress response, ROS production and fusaric acid production 73 suggesting that VIP2 controls several physiological and metabolic pathways. Modulating VIP2 expression might be a useful tool for enhancing plant transformation efficiency in plant species where endogenous VIP2 expression is at low level. However, susceptibility of VIP2 overexpressor lines to ABA and other abiotic stresses (Fig. 7 ) can be an issue. It requires further studies to understand how VIP2 regulates abiotic stress responses.
Methods Yeast one-hybrid assay. The yeast one-hybrid assay was performed as described earlier 24 . VIP2 (GenBank # AF295433) open reading frame was fused to the GAL4 DNA binding domain (BD) and cloned into a pGBKT7 yeast vector containing the auxotrophic marker trp1 gene and the GAL4 upstream activating sequence (UAS promoter). The human lamin C and topoisomerase I genes cloned into the pGBKT7 vector were used as controls. These two genes are known to function as non-specific activators of the auxotrophic marker, but can be deactivated by the addition of 3-amino-1,2,4-triazole (3-AT). All clones were transformed into the MaV204K yeast strain. A single colony of each transformed MaV204K was picked and grown overnight in 100 μl liquid synthetic dropout (SD) medium lacking tryptophan in 96 well plates. All cultures were transferred into a new 96 well plate with three different dilutions of the liquid culture (1:10, 1:100 and 1:1000). Cultures were plated onto solid SD medium, lacking tryptophan and containing 3-AT, using 96-well pin replicator, and incubated at 30 C overnight for the single-dropout assay. The three clones fused to the GAL4-BD were also replicated on SD medium lacking tryptophan and histidine, and containing 10 mM 3-AT for the double-dropout assay. transactivation analysis. A promoter containing three copies of the GAL4 upstream activating sequence (UAS), and VIP2 were fused to the DBD of GAL4 modified for optimal expression in plants (mGAL4) and expressed constitutively under the CaMV35S promoter from a pSAT6 vector (pSAT6-mGAL4-DBD-VIP2). Additionally, the pSAT6-uasP-GUS vector 25 with GUS reporter gene fused to the minimal UAS promoter (uasP) was also included as a control. The biolistic approach for DNA delivery was performed using standard protocols and plasmids as described 25 . Bait and prey construction. The full-length, C-terminal, and N-terminal of VIP2 and the VirE2 genes were PCR amplified using a high-fidelity platinum Pfx DNA polymerase (Invitrogen Inc.) and cloned into the pENTR/D Topo vector (Invitrogen, Carlsbad, CA). All these clones were then cloned into the yeast two-hybrid DNA BD vector pXDGAT-CY86 with the cyclohexamide sensitive gene (CYH S ) 74 . The full-length VIP2 and VirE2 genes were also cloned into the DNA activation domain (AD) domain vector, pGADT7-Rec7G to be used as preys. The prey vector pGADT7-Rec7G was constructed from the pGADT7-Rec7 vector (Clontech, Mountain View, CA) by introducing the GATEWAY cassette at the SmaI site using standard cloning protocols and confirmed by sequencing. Recombination between pENTR/D Topo vectors and the destination vector was performed using the Clonase II enzyme mix according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Bait and prey constructs were confirmed to be in-frame with the DNA-BD or DNA-AD prior to transformations. Bait constructs were transformed into the MaV204K yeast strain (MAT α, leu2-3,112; trp1-901; his3 Δ200; ade2-101; cyh2 R ; can1 R ; gal4 Δ; gal80 Δ; GAL1::lacZ;HIS3UASGAL1::HIS3@LYS2; SPAL10::URA3) 75 . Prey constructs were transformed into AH109 yeast strain (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4 Δ, gal80 Δ, LYS2 ::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1). All transformations were performed as described 76 . All bait strains were checked for auto activation of the reporter gene, his, by checking their growth on SD -His/-Trp supplemented with 0, 2.5, 5, 7.5, 10 and 15 mM 3-AT. The 3-AT concentration of 10 mM was found to inhibit auto activation of the both bait or prey clones, so it was used as media supplement in the rest of the study. Mapping the interaction between VirE2 with VIP2. Pairs of various bait and prey constructs were co-transformed into AH109 strain to test the specificity and to map the interaction between VirE2 and VIP2 (Fig. 2a ). Co-transformants were selected on solid SD medium lacking Ade, His, Trp, and Leu and supplemented with 10 mM 3-AT and X-gal. Positive clones were then re-grown in SD liquid medium under the same selection conditions. The selection was repeated four times by sub culturing positive clones and assayed for the activity of X-gal. Clones that maintained an ability to grow under selected conditions were then grown on liquid SD medium in a 96-well (Fig. 2a , left panel) plate along with yeast strains containing only bait or prey constructs as negative controls (Fig. 2a , right panel). Validation and β-galactosidase assays. Co-transformed yeast strains with various VIP2 and VirE2 constructs, and only bait or prey, were grown on 96 well plates containing liquid SD medium that lacks Ade, His, Trp, and Leu supplemented with 10 mM 3-AT for 24 h. Absorbance at A 600 was measured for three independent 100 μl cultures and then assayed for β-galactosidase activity using the yeast β-galactosidase assay kit (cat. 75768; Pierce Biotechnology, Inc.) following the manufacturer's instructions. A 600 and A 420 were measured using multi-plate reader, Tecan infinite 200Pro. The β-galactosidase activity was calculated using the equation: β-galactosidase activity = 1000 × A 420 /T × V × A 600 where T is time (in minutes) of incubation and V is volume of cells (ml) used in the assay. plant transformation experiments. We tested whether constitutive overexpression of AtVIP2 in the Atvip2 mutant and Col-0 plants could restore the wild-type phenotype and/or increase the transformation efficiency. Briefly, for over-expression studies, cDNA corresponding to the Arabidopsis VIP2 cDNA was amplified by RT-PCR along with GATEWAY adapter primers, sequence verified and cloned into the plant expression vector pMDC32 77 driven by constitutive CaMV35S promoter. Wild-type Arabidopsis Col-0 and Atvip2 mutant plants were transformed by the floral dip method 78 . The transgenic plants were selected for resistance to hygromycin and also tested for the presence of the transgene using the GATEWAY adapter primers (attB1/B2), while the expression of the transgene was confirmed by semi-quantitative RT-PCR using gene specific primers. These transgenic plants were selfed and T1 homozygous seeds were collected.
Root tumor, GUS and callus assays. Arabidopsis root tumor assays were performed as described earlier 15, 28, 30 . For root tumor assays, axenic root segments from wild-type Col-0 and transgenic plants were infected with a tumor-inducing A. tumefaciens strain, native A208 containing the nopaline type Ti-plasmid pTiT3, co-cultivated for 48 h in the dark at room temperature and transferred to a hormone-free Murashige and Skoog media (MS) supplemented with cefotaxime (250 mg/l) and timentin (100 mg/l). Tumor numbers and phenotypes were recorded at 4 weeks after infection. Stable GUS transformation assays were performed as described earlier 27, 39 . Root segments from Col-0 and transgenic plants were infected with disarmed strain of A. tumefaciens GV3101 carrying uidA-intron gene within the T-DNA and co-cultivated for 48 h at room temperature. The root segments were incubated on callus induction medium (CIM) for 2-3 weeks, and stained with X-gluc. Stable transformation callus assays were carried out as described earlier 28, 30 . Root segments from wild-type Col-0 and transgenic plants were infected with a disarmed A. tumefaciens strain GV3101 containing pCAS1 28 and co-cultivated for 48 h in dark at room temperature. The root segments were incubated on CIM supplemented with phosphinothricin (PPT) at 10 mg/l, cefotaxime (250 mg/l) and timentin (100 mg/l). The number of root segments forming PPT-resistant calli was counted at 4 weeks after infection.
Tissue specific expression of VIP2. The promoter sequence upstream of the ORF (1 kb region, see ppdb: Plant promoter database, http://ppdb.agr.gifu-u.ac.jp/ppdb/cgi-bin/index.cgi, Fig. 4b ) 79 was PCR amplified, confirmed by sequencing, and fused to a reporter gene (uidA) in the pMDC162 vector, and mobilized into A. tumefaciens strain GV3101. Arabidopsis plants were transformed with the VIP2 Promoter:uidA fusion expression cassette via the floral dip transformation method and hygromycin-resistance transgenic T1 events were identified for promoter expression analysis. Three to four independent T1 lines were selected and screened by histochemical staining for GUS expression. Microarray gene expression analysis. Three biological replicates were performed for each tissue sample. Total RNA was isolated as described previously 15 . RNA was quantified and evaluated for purity using a Nanodrop Spectrophotometer ND-100 (NanoDrop Technologies, Willington, DE) and Bioanalyzer 2100 (Agilent, Santa Clara, CA). Ten μg of total RNA was used for the expression analysis of each sample using the Arabidopsis ATH1 chip (Affymetrix, Santa Clara, CA). Probe labeling, chip hybridization and scanning were performed per the manufacturer's instructions for one-cycle labeling (Affymetrix). Data normalization between chips was conducted using RMA (Robust Multichip Average) 80 . Presence/absence calls for each probe set were obtained using dCHIP 81 . Gene selections based on Associative T-test 82 were made using Matlab (MathWorks, Natick, MA). In this method, the background noise present between replicates and technical noise during microarray experiments was measured by the residual present among a group of genes whose residuals are homoscedastic. Genes whose residuals between the compared sample pairs that are significantly higher than the measured background noise level were considered to be differentially expressed. A selection threshold of 2 for transcript ratios and a Bonferroni-corrected P value threshold of 2.19202E-06 were used. The Bonferroni-corrected P value threshold was derived from 0.05/N in these analyses, where N is the number of probes sets (22,810) on the chip. Functional classification of genes was carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 83 with the Benjamini correction and false discovery rate (FDR) <0.05, as well as MAPMAN (http://mapman.gabipd.org/) using log 2 transformed value of transcript ratios. In silico motif analysis. The 500 bp upstream region of one hundred genes from each class was downloaded from RSAT site (http://floresta.eead.csic.es/rsat/) with the option preventing overlap with neighbor genes (noorf). Promoters were analyzed using the online tool MotifSampler (http://bioinformatics.intec.ugent.be/MotifSuite/ motifsampler.php). A precompiled background model of Arabidopsis with order set to 3 was used. The total number of runs was set to 50. MotifRanking (http://bioinformatics.intec.ugent.be/MotifSuite/motifranking.php) was used to rank highest scoring motifs based on LogLikelihood ratios.
ABA quantification. Freshly harvested plant tissues were frozen in liquid nitrogen and ground to a fine powder, and stored at -80 C until ABA extraction. ABA quantification was carried out as described 84 . In brief, 1 ml of cold methanol:water (70:30, v-v) plus labeled ABA was added to 100 mg of powdered tissue. Samples were vortexed, sonicated, and extracted at 4 C for 1 h. Samples were centrifuged at 16,000 × g for 5 min at 4 C and the supernatants were dried using nitrogen gas. The residues were re-dissolved in 100% methanol, and the supernatant injected into an Agilent 1290 UHPLC connected to an Agilent 6430 Triple Quad mass spectrometer (Agilent Technologies). Separation was carried out using a Waters BEH C18 column (Waters Co., 1.76 μm, 2.1 × 150 mm). The following solvents were used at a flow rate of 0.4 ml min -1 : (A) 0.05% formic acid/H 2 O and (B) acetonitrile/0.05% formic acid. The separation was achieved by starting with 5% solvent B, a gradient from 5% to 46% of solvent B over 19 min and a step to 90% B in 0.1 min, then a hold at 90% B for 2 min and a step to 5% solvent B in 0.1 min. The temperature of the UPLC column was set to 40 C. The gas temperature was 300 C, gas flow: 9 ml/ min, nebulizer was 25 psi. Fragmentor and collision energy were optimized for each compound individually. The SRM analysis conditions for ABA and d 6 ABA (negative ion mode) were as follows: capillary = 4,000 V, fragmentor voltage = 100 V, collision energy = 4 V, dwell time = 200 ms and SRM transition (m/z) = 263/153 for unlabeled ABA and 269/159 for d 6 ABA. Relative amounts of ABA were based on comparison to the labeled hormone. Abiotic stress experiments. Seeds of Arabidopsis ecotype Col-0 were surface sterilized in 75% ethanol for 2 min, and 30% bleach for 15 min, followed by four washes with sterile water. The seeds were sown onto half-strength MS medium containing 1% sucrose and 0.3% phytagel, stratified in the dark at 4 C for 2 d and grown in growth chamber with 12.5 h day length at 24 C. Seven days after stratification, seedlings were transferred to half-strength MS plates containing 5 μM ABA, 10 μM ABA, 75 mM mannitol, and 100 mM NaCl. Fifteen days after transfer, fresh weights of the plants were measured. Figure 1 . 1 Figure 1. Yeast one-hybrid assays and in planta transactivation assays suggest VIP2 is a putative transcription regulator. (a) Single and double dropout assays were carried out on SD medium lacking Trp, or Trp and His respectively, and containing 10 mM 3-AT. Undiluted, and dilutions of 1:10, 1:100 and 1:1000 were plated. The experiments were repeated twice. (b) A constitutive CaMV35S promoter driving VIP2 was fused to the DNA binding domain (DBD) of a GAL4/UAS transactivation expression system in combination with the GUS reporter protein. GUS histochemical staining of tobacco leaves bombarded with different reporter constructs pSAT6-uasP-GUS only, pSAT6-uasP-GUS and pSAT6-mGAL4-DBD constructs, and pSAT6-uasP-GUS and pSAT6-mGAL4-DBD-VIP2 constructs were carried out. The experiments were repeated three times with similar results.
Figure 2 . 2 Figure 2. Yeast-two hybrid assay suggests the C-terminal of VIP2 protein interacts with VirE2. These various domains of VIP2 and full length VirE2 were cloned into yeast two-hybrid vectors pXDGAT-CY86 and pGADT7, respectively, and were independently transformed into AH109 and MaV204K yeast strains, followed by mating and selection in triple dropout media. (a) The LacZ expression in the various bait and bait/prey interactions containing either the full length proteins or the different VIP2 domains. (b) Quantitative LacZ activities between various haploid or diploid clones were further confirmed. The experiments were repeated twice with similar results.
Figure 3 . 3 Figure 3. Complementation of the Atvip2 mutant by constitutive expression of AtVIP2. Three independent transgenic events were generated, and molecularly characterized before performing root transformation assays. (a) Roots of the wild-type, vip2 mutant and three transgenic Atvip2 lines expressing AtVIP2 cDNA were infected with a tumorigenic strain A. tumefaciens A208, at 1 × 10 7 CFU/ml concentration. Tumors incited on the roots were visualized and scored 4 weeks after infection. (b) Stable GUS expression. Roots of the wild-type, Atvip2 mutants and transgenic Atvip2 lines expressing AtVIP2 cDNA were inoculated with A. tumefaciens strain GV3101 carrying the uidA-intron gene within the T-DNA at 1 × 10 7 CFU/ml concentration. The inoculated roots were stained with X-Gluc 2-3 weeks post infection. All the experiments were repeated two times with similar results.
Figure 4 . 4 Figure 4. AtVIP2 gene expression and the AtVIP2 promoter region. (a) The amplification of AtVIP2 transcripts from different plant organs (leaves, stem, root, petioles and flower) in Arabidopsis by semi-quantitative PCR. NC indicates negative control. (b) Schematic presentation of the different regulatory elements on the 1 kb promoter sequence identified upstream of the AtVIP2 coding sequence as shown in plant promoter database (http://ppdb.agr.gifu-u.ac.jp/ppdb/cgi-bin/index.cgi). (c) Analysis of Arabidopsis whole seedlings expressing GUS gene under the control of AtVIP2 promoter. Histochemical GUS staining of transgenic Arabidopsis whole seedlings; roots and root hairs; leaf; trichrome, stem, and floral organs. Pictures were taken 3-4 days after GUS staining.
Figure 5 . 5 Figure 5. Transcript profiling of AtVIP2 overexpressor plants during different stages of Agrobacterium infection. (a) Venn-diagram showing overlap between differentially regulated (DE) genes, compared to Col-0, in AtVIP2 overexpressor plants during different time points after Agrobacterium infection. (b) GO term enrichment for up-regulated and down-regulated genes from all time points using Database for Annotation, Visualization and Integrated Discovery.
Figure 6 . 6 Figure 6. ABA content of wild-type, Atvip2 mutant and AtVIP2 overexpression lines. Three weeks old seedlings grown in half MS media were used for ABA quantification. Error bars indicate SE of the mean (n >= 8). Asterisks indicate significant differences (*P <= 0.05 and P <= 0.005) between Col-0 and other plants, as determined by two-way ANOVA, uncorrected Fisher's LSD test.
Figure 7 . 7 Figure 7. Effect of abiotic stress conditions on the growth of wild-type, Atvip2 and AtVIP2 overexpressor plants. Growth of plants under (a) normal (half strength MS), (b) 5 μM ABA, (c) 10 μM ABA, (d) 75 mM mannitol and (e) 100 mM NaCl. In all graphs, error bars indicate SE of the mean (n >= 8). Asterisks indicate significant differences (*P <= 0.05 and P <= 0.005) between Col-0 and other plants, as determined by two-way ANOVA, uncorrected Fisher's LSD test.
Scientific RepoRtS | (2019) 9:13503 | https://doi.org/10.1038/s41598-019-49590-3
|
10.3390/s7112741
|
cc-by
| null | null |
openalex
|
A very compact (device area around 40 μm 2 ) optical ammonia sensor based on a microring resonator is presented in this work. Silicon-on-insulator technology is used in sensor design and a dye doped polymer is adopted as sensing material. The sensor exhibits a very good linearity and a minimum detectable refractive index shift of sensing material as low as 8x10 -5 , with a detection limit around 4 ‰.Introduction Ammonia (NH 3 ) concentration measurement has a great importance in many scientific and technological areas. In environmental monitoring, automotive and chemical industry, electronic and optical ammonia sensors are widely used . Recently the possibility to diagnose by ammonia sensing certain diseases, as ulcer or kidney disorder, has been proved. For example, NH 3 concentration level measurement in exhaled air is a fast and non-invasive method to detect the presence of Helicobacter pylori bacterial stomach infection . The most frequently used technique in commercial ammonia detectors is based on SnO 2 and MoO 3 semiconductor thin films. These sensors are mainly used in combustion gas detectors or gas alarm systems, but they show some limitations in reproducibility, stability, sensitivity and selectivity. Other approaches to NH 3 sensing are based on potentiometer electrodes , infrared gas analyzers and conducting polymers . Different kinds of optical ammonia concentration sensors, based on monitoring the absorption or fluorescence characteristics of sensing films deposited onto an optical fiber, have been reported . An integrated optical ammonia sensor based on a Y-junction has been reported in . This sensor employs a deposited sensing film whose absorbance (dependent on ammonia concentration in the surrounding ambient) is continuously measured and exhibits a detection limit around 1ppm. Ammonia concentration estimation by this sensor implies an optical power differential measure at the output of the two Y-junction arms. In recent years, integrated optical sensors have attracted considerable attention because of their immunity to electromagnetic interference, high sensitivity, good compactness and robustness and high compatibility with fiber networks . A great variety of guided-wave optical sensors has been proposed, such as those based on directional couplers , Mach-Zehnder interferometers , grating-assisted couplers , and optical microcavities . In particular, optical microring resonators, widely used in add-drop filters, optical switches, ring lasers and WDM multiplexers, are showing very attractive features for sensing applications, permitting to realize highly sensitive immunochemical optical biosensors . In this paper we design, optimize and 3D simulate an integrated optical microring resonator-based ammonia sensor. Device sensitivity dependence on waveguide optical and geometrical parameters is investigated. Sensor detection limit is also analyzed.
Sensing Principle The architecture of a very compact microring resonator-based sensor in Silicon on Insulator (SOI) technology is sketched in Figure 1 (a). A ridge structure has been adopted as waveguide (this kind of sub-micrometer guiding structure is also indicated as silicon photonic wire), as in Figure 1 Optical absorption changes of PMMA-BCP system, due to interaction with ammonia, has been proved either when PMMA-BCP is exposed to dry ammonia (in this case PMMA-BCP sample is put in a chamber filled by ammonia diluted with pure nitrogen to a molar concentration of 5%) or when PMMA-BCP is exposed to a vapor of conventional medical ammonia spirit (65% alcohol). This optical absorption change produces a shift in polymer refractive index at wavelengths outside the material absorption band, according with the Kramers-Krönig relationship: ( ) ( ) 2 2 0 PMMA BCP PMMA BCP c n p d Δα Λ Δ λ Λ π λ Λ ∞ - - = - ∫ ( 1 ) where Δn is the PMMA-BCP refractive index change, λ is the wavelength at which Δn is calculated, c is the light speed in vacuum, p stands for the principal value of the integral, and Δα is the PMMA-BCP absorption coefficient change. Since PMMA-BCP absorption band extends from 350 nm to 450 nm, then polymer interaction with NH 3 causes a refractive index shift at wavelengths around 1550 nm, given by : ( ) ( ) ( ) 0 0 PMMA BCP t b n , C F , vS p C Δ λ λλ ε λ - = (2) where λ 0 is the absorption band central wavelength, F(λ, λ 0 ) is the proportionality factor between absorption and index changes, v t is the total number of BCP molecules per PMMA unit of volume, S b is the reagent-analyte binding constant, p is the polymer permeability factor and C is the ammonia ambient concentration. Thus, a PMMA-BCP refractive index change determinates an effective index N change of mode propagating around the ring resonator. Microring resonant wavelength λ R is related to effective index by the following relation: R NL m λ = (3) where m is the resonant mode order (m is an integer number) and L is the ring resonator length. Effective index change implies a microring resonant wavelength shift (as schematically reported in Figure 2 ). Measuring the resonance wavelength shift around 1550 nm, it is possible to estimate the ammonia concentration in gaseous medium surrounding the sensor. The adoption of wavelengths around 1550 nm for optical sensing allows the additional advantage to use standard telecom equipments as lasers, photodetectors and so on.
Device Sensitivity The device sensitivity is one of the most important and critical aspect in sensor design and its theoretical estimation depends on a number of device geometrical and physical parameters. Sensitivity of the designed sensor can be expressed as: R R c c N n N n λ λ Δ Δ Δ = Δ Δ Δ (4) where λ R is around 1550 nm and whose shift is measured to estimate the ammonia concentration, N is the ring mode effective index and n c is the cladding layer refractive index. Variation rate of N (as ΔN/Δn c ) has been numerically estimated by a commercial mode solver for bent waveguides based on Alternating Direction Implicit (ADI) method, by varying n c in a narrow range and observing the corresponding shift of N. The resonance wavelength incremental change (as a ratio between Δλ R and ΔN) has been analytically estimated using the approach proposed in , obtaining the following formula: ( ) 1 0 , R c R R R N n N N λ λ λ λ λ λ - = ⎡ ⎤ Δ ∂ ⎢ ⎥ = - Δ ∂ ⎢ ⎥ ⎣ ⎦ (5) where n c 0 is the PMMA-BMC refractive index before any interaction with ammonia. In equation (5) , N(n c 0 , λ r ) and its derivative with respect to λ have been numerically estimated by ADI method. By Eq. (5), it is possible to obtain the relationship: ( ) 1 0 , R c R R c c R N n N N n n λ λ λ λ λ λ - = ⎡ ⎤ Δ Δ ∂ ⎢ ⎥ = - Δ Δ ∂ ⎢ ⎥ ⎣ ⎦ (6) useful to make accurate estimations of the proposed sensor sensitivity. Thus, we have investigated the device sensitivity dependence on ridge height (h) and width (w), for different values of microring resonator radius (R) (see Figure 3 and 4 ). It can be observed the sensitivity as nearly independent from R, whereas it exhibits a quadratic dependence on w and h. Moreover, it depends more strongly on ridge height than on its width. To find optical and geometrical parameters affecting the device sensitivity, we have investigated how all quantities in formula (6) are influenced by waveguide electromagnetic properties. According with variational theorem for dielectric waveguides, we can write: 0 2 0 2 c c cladding n N dxdy n P η Δ = Δ ∫∫ E (7) ( ) 2 2 2 1 0 0 1 , R R N n x y dxdy P λ λ η η λ λ - = ∞ ⎡ ⎤ ∂ = - + ⎢ ⎥ ∂ ⎣ ⎦ ∫∫ E H ( 8 ) where P is the integral of time-averaged Poynting vector component along the propagation direction z,, n(x,y) is the waveguide refractive index profile, and η 0 is the free space impedance. By using ( 6 ), ( 7 ) and ( 8 ), the device sensitivity can be written as: ( ) 2 0 2 0 2 0 2 , c R cladding R c c R core core n d x d y n PN n n dxdy λ λ η λ Δ ≅ Δ + ∫∫ ∫∫ E E (9) where n core is the core layer refractive index and some additive contributions at denominator can be neglected. Relationship (9) shows how sensitivity strongly depends on the index contrast between cladding and core layers. By decreasing this contrast, it is possible to improve the sensitivity, but this strategy implies an increase of microring radius to avoid too large bend radiation losses.
Sensor Design and Simulation In our design we have chosen a 300 nm high and 250 nm width ridge waveguide and a microring radius of 3 μm. The optical sensor has been 3D simulated using Finite-Difference Time-Domain (FDTD) technique . Waveguide modal behavior has been investigated and a single-mode condition has been satisfied (see profile of quasi-TM single mode in Figure 5 ). In absence of any change of cladding refractive index, optical propagation in the microring has been simulated as in Figure 6 , and Discrete Fourier Transform (DFT) of electric field y component has been calculated at the output cross section (see Figure 7 ). When no cladding interaction with ammonia occurs, the ring exhibits a resonance wavelength at 1545.33 nm. Then, shift from this resonance wavelength due to PMMA-BCP refractive index change has been calculated. Since ammonia diluted with pure nitrogen to a molar concentration of 5% should induce a PMMA-BCP refractive index change around 10 -3 , the resonance wavelength shift produced by cladding refractive index shift varying from 1x10 -3 to 9x10 -3 has been sketched, as in Figure 8 . A very good linearity can be observed. Proposed sensor sensitivity, as Δλ R /Δn c , has been calculated by 3D FDTD simulations, obtaining a value of 132 nm. This value exhibits a good agreement with that estimated by formula (6) . Since microring resonance wavelengths are more than 20 nm spaced whereas maximum resonance wavelength shift over the sensor operating range is below 2 nm, the minimal detectable cladding refractive index shift also depends on accuracy in estimating the resonance wavelength shift. If a tunable laser (operating around 1550 nm) and a photodetector are used to this aim, a minimum detectable wavelength shift of 10 pm should be obtainable. Therefore, a minimum detectable cladding refractive index shift below 8x10 -5 should be achievable by our approach. Minimum detectable ammonia concentration change is then estimated as around 4 ‰. This detection limit value is significantly less accurate than that reported for other guided-wave optical ammonia sensors (see, for example, that reported in ). However, our SOI integrated sensor approach is significantly more compact (device area around 40 μm 2 ). Moreover, since our sensor readout technique is based on the estimation of a wavelength shift (wavelength interrogation), it is intrinsically more immune to optical noise than any other integrated sensing device where the readout technique is based on amplitude estimation or a comparison of optical powers (amplitude interrogation). Finally, adoption of SOI technology permits the full integration of the optical/electronic readout circuit on the same sensor chip to be realized. Finally, device sensitivity to ammonia concentration change is limited by the temperature influence on cladding refractive index. It has been already demonstrated that each degree of temperature shift produces a change in PMMA refractive index of about 1x10 -5 . Therefore, it is necessary to keep constant the gas temperature with high accuracy in order to detect an ammonia concentration change that produces a cladding refractive index shift lower than 8x10 -5 .
Conclusions We have designed, simulated and optimized a very compact (about 40 μm 2 ) integrated optical ammonia sensor. Polymethylmethacrylate doped with Bromocresol Purple has been adopted as sensing material because its refractive index around 1550 nm changes after appropriate interaction with NH 3 . Proposed device exhibits a very good linearity over a wide range, a sensitivity of 132 nm and it is able to reveal a sensing layer refractive index change as low as 8x10 -5 and an ammonia concentration change around 4 ‰. (b), and Polymethylmethacrylate (PMMA) doped with Bromocresol Purple (BCP) has been used as cladding layer.
Figure 1 . 1 Figure 1. (a) Ammonia sensor architecture. (b) Ridge guiding structure adopted in sensor design.
Figure 2 . 2 Figure 2. Proposed ammonia sensor operating principle.
Figure 3 . 3 Figure 3. Device sensitivity versus ridge height, numerically estimated by formula (6) (quasi-TM mode).
Figure 4 . 4 Figure 4. Device sensitivity versus ridge width, numerically estimated by formula (6) (quasi-TM mode).
Figure 5 . 5 Figure 5. Quasi-TM mode propagating in proposed sensor waveguide.
Figure 6 . 6 Figure 6. Optical propagation in microring sensor in absence of any change of cladding refractive index.
Figure 7 . 7 Figure 7. Field component E y DFT at output cross section.
Figure 8 . 8 Figure 8. Shift in resonance wavelength versus shift in cladding layer refractive index.
2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
|
10.21123/bsj.16.1.(suppl.).0194
|
cc-by
| null | null |
openalex
|
A simple, rapid spectrophotometric method has been established for the determination of chlorpromazine hydrochloride (CPZ) in its pure form and in a tablet formulations. The suggested method is based on the oxidative coupling reaction with4-nitroainlline using KIO 3 in acidic solution to produce a violet colored product with maximum absorption at λ=526 nm.The analytical data obtained throughout this study could be summarid as follows: 1ml of 1M HCl (pH=2.2), 1 ml of 4-nitroanilline (1x10 -2 M), and 1.5ml of (1x10 -2 )KIO 3 per 25 ml reaction medium. The order of additions, coupling reaction time, and temperature in addition to the type of solvent were studied. The Beer′s law is obeyed over the concentration range of(5-40) μg ml -1 , but the detection limit and quantification limit are 0.34 besides 1.03 μg ml -1 respectively. The correlation coefficient (r) for the calibration graph was found to be 0.9980, molar absorptivity of 10. 25 × 10 3 L.mol -1.cm -1 , and Sandell′s sensitivity index of 0.03467 μg.cm -2 . The precision and accuracy of the method were tested by calculating the percentage of relative standard deviation (RSD%) (<1.831%) and the average recovery percent (99.22%) average error percent E rel% (0.558). Direct and standard addition procedures were applied to both standards and specimens of pharmaceutical and the results indicate that the suggested method was successfully applied for the determination of CPZ.Introduction: Chlorpromazine Hydrochloried (CPZ) is the most important compound in the group of phenothiazine derivatives. CPZ is widely used as a healing agent for treating various psychological and personality illnesses (1) (2) (3) . It is 3-(2-chloro-10H-phenothiazin-10yl)-N,N-dimethylpropan-1-aminehydrochloride C 17 H 19 ClN 2 S.HCl (Fig. 1 ). Like other phenothiazine derivatives it contains heterogeneous rings containing sulfur and nitrogen atoms. Department of Chemistry, College of Education for pure sciences, University of Tikrit, Tikrit, Iraq. * Corresponding author: asmamohahh@tu.edu.iq A number of methods have been applied for the determination of chlorpromazine hydrochloride in pure and dosages. These include HPLC (4-5), liquid-mass spectroscopy (6) , potentiometry (7) (8) (9) , LC-MS/MS (10) , GC (11) , flow injection analysis (12) (13) , voltammetry (9), Among the spectrophotometric methods, several methods were proposed depending upon the oxidation of drug using various oxidizing agents (14) (15) (16) (17) . In this work, a study of the spectrophotometric determination of CPZ-HCl relying on the oxidative coupling of the drug with 4-nitroaniline using potassium iodate as oxidation reagent in acidic medium.
Materials and Methods: T90 UV-Visible Spectrophotometer PG Instrumental Ltd, UK with 10 mm quartz cell was used for all spectrophotometric quantities, Jenway 3310 pH meter was used to check the pH of solutions, and Sartorius Balance 210S kern was used to perform all weight measurements.
Reagents Fully chemicals used were of analytical grade and chlorpromazine. HCl typical material was provided from State Company for Drug Industries and Medical Appliance (SDI) Samarra-Iraq. Distilled water was used to prepare all solutions. A 1000 μg/ml (2.814 × 10 -3 M))CPZ of solution was prepared by dissolving 0.100gm in100ml distilled water and stored in dark and used, for at least one month, as stock solution. More dilute working solutions of the drug were prepared by serial dilutions with distilled water. 4-nitoanilline solution (1.000 × 10 -2 M ) was freshly ready daily by dissolving0.0345gm in 5ml ethanol and completed by distilled water to 25ml , while the calculated weight of KIO 3 0.2140gm dissolved in 100ml distilled water was required to prepare (1.000 × 10 -2 M) solution. 1.000 × 10 -1 M solution of each of sodium hydroxide and hydrochloric acid was prepared and used.
General procedure In a 25 ml volumetric flask, 1 ml of 1.000 × 10 -2 M 4-nitroanilline (R) solution is mixed with 1.500 ml of 1.000x10 -2 M KIO 3 . To this mixture 3 ml aliquot of CPZ solution is added and the mixture is rendered acidic by the addition of 1. 000ml of 1.000 M HCl (11.8N). The mixture is then diluted and the value of absorbance is measured at 526 nm after letting the mixture to stand for 15 minutes in the dark.
Application to various dosage forms A portion equivalent to 0.1075 gm of a homogenized powder of 10 tablets Largactil (Aubrey Pharmaceutical Industries -Aleppo/Syria with distinction from the company Avnitis -France 25mg/tablet) was weighed and dissolved in 100 ml water. Dissolution of the drug was assisted by means of magnetic stirring and an ultrasonic bath. The resulted solution was cleaned through a Whatman filter paper No.1. and the volume was made up to the dent with distilled water in 100ml calibrated flask. The drug gratified of an aliquot of this solution was found by applying the general procedure as pronounced above.
Results and Discussion: When CPZ was treated according to the recommended procedure, the recorded absorption spectrum of the formed reaction product for the range of 450 to 600 nm against reagent blank shows an extreme absorption at 526 nm, while, the blank does not have any significant absorbance in this region, as it is shown in (Fig. 2 ).
Optimization of reaction conditions: The effect of the amount of HCl, 4-nitroanilline, and potassium periodate was investigated. The results show that use of 1.000 ml of 1.000 M hydrochloric acid (i.e. pH=2.2) with 1.500ml of 1.000 × 10 -2 M of KIO 3 , and 1ml of 1.000 × 10 -2 M of coupling reagent give best results (Fig. 3 a, b and c respectively). The order of addition of the reactants should be followed, as cited in the recommended procedure. Moreover, optimum time for full color development was found to be 10minutes, and room temperature15C ο was found the most favorable for the color development of the reaction product, Fig. 4 (a and b respectively). Furthermore, water was found to be the best solvent among different solvents (viz: water, ethanol, acetone and diethyl ether) tried to solvate the reaction medium and to obtain the perfect absorbance, Fig. 5 .
Calibration graph and the statistical data Using the obtained optimum conditions, a calibration curve was built (Fig. 6 ). The graph shows that the violet colored product obeys Beer's law in the range of concentration of 5 -40 μg/ml of CPZ. Table 1 .depicts the statistical information of the calibration curve of spectrophotometric determination of CPZ.
precision and Accuracy The capability of the method was statistically evaluated via measuring accuracy as relative error percentage (E rel %), and precision as relative standard deviation percent of the proposed methods. Table 2 illustrates that the results obtained for seven replicates at three concentration levels of CPZ were satisfactory which indicate that the proposed methods have a good precision and accuracy.
Table2. Evaluation of precision and accuracy for determination of CPZ . *
* n=7
Stoichiometry of reaction The stoichiometry of the response between CPZ and the mixture was investigated using Job's method and mole-ratio method. The consequences obtained via the two methods indicate that the stoichiometry of the water-soluble coupling product between drug and the reagent is 1:1 (Fig. 7 and Fig. 8 )(15). Accordingly, a mechanism for formation of the colored product upon the reaction between CPZ and reagent in presence KIO 3 is founded in scheme 1: Scheme 1. The suggested mechanism for formation of the colored product.
Analysis of CPZ in pharmaceutical preparations Two procedures (direct calibration and standard additions) using the proposed method to determine CPZ in Largactil tablets. The obtained results are shown in Table 3 and in Fig. 9 . Good agreement in results was found for both procedures.
Conclusion: A rapid, simple and precise spectrophotometric method has been suggested for the determination of chlorpromazine hydrochloride in aqueous solution based on oxidation with 4-nitroanilline and KIO 3 in the acidic solution. The suggested method does not require temperature control or the solvent extraction step, the method was applied, successfully for the determined of amounts commercial CPZ drug. Figure 1 . 1 Figure 1. The structure of chlorpromazine hydrochloride.
Figure 2 . 2 Figure 2. The absorption spectra of 30 μg/ml CPZ-R coupling product against reagent blank solution, 30 μg/ml CPZ -R coupling product against distilled water, and reagent blank solution against distilled water.
Figure 3 . 3 Figure 3. Effect of (a) HCl, (b) KIO 3 , and (c) 4nitroanaline on the on the formation of the colored product.
Vol. of 1M HCl (ml) (b)Vol.of 0.01M KIO3 (ml) (c)Vol.of 0.01M 4-nitroanilline (ml)
Figure 4 . 4 Figure 4. Effect of (a) coupling time, (b) Temperature, on the formation of the colored product.
Figure 5 . 5 Figure 5. Effect of solvent.
Figure 6 . 6 Figure 6. Calibration curve for the determination of chlorpromazine hydrochloride under optimal condition.
Figure 7 . 7 Figure 7. Job's method.
Figure 8 . 8 Figure 8. Mole ratio for reaction of (CPZ) with reagent.
Figure 9 . 9 Figure 9. Standard additions plot.
Table 1 . Statistical data of the calibration curve for the spectrophotometric determination of CPZ. 1 Parameter Value λ max nm 526 Color Violet Linear range (μg/mL) 5-40 Regression equation A = 0.0298 [CPZ] + Molar absorptivity (L/mol.cm) 0.002 1.025×10 4 Correlation coefficient 0.9980 Intercept 0.002 Slope 0.0298 S b 0.0307 S a 0.197 LOD (μg/mL)(18) 0.0930 LOQ (μg/mL)(18) 1.03 Sandell's sensitivity (μg/cm 2 ) 0.03467
Table 3 . Determination CPZ in Largactil Tablets (25 mg/tablet), by direct calibration and standard addition method. 3 Procedure Conc. of CPZ (μg/ml) AE SD RSD% E rel % Recovery % Taken Found Direct calibration. 10 10.06 0.06 1.41 0.009 0.68 100.68 40 40.27 0.27 1.45 0.360 0.675 100.675 Standard additions 25 23.94 -1.06 1.42 0.589 -4.219 95.780
|
10.5281/zenodo.7496189
|
cc-by
| null | null |
openalex
|
The main forms of periodontal disease are superficial lesions of the gums -gingivitis and deeper -periodontitis. Periodontitis has synonyms: alveolar pyorrhea, periodontitis, periodontosis, periodontoclasia, alveoloclasia, Fauchard's disease, etc. In 1983, the plenum of the All-Union Scientific Society of Dentists adopted a classification that is currently widely used in the CIS countries. In accordance with it, the initial form of periodontitis is gingivitis, which is an inflammation of the gums, caused by the adverse effects of general and local factors, occurring without violating the integrity of the gingival junction. The main role in the occurrence of periodontal diseases is played by microorganisms included in the "red complex" BANA +: Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia. With the simultaneous identification of these types, one can judge a high risk of developing periodontal diseases. Purpose -to determine the frequency of occurrence of various types and topography of dentition defects in patients with varying degrees of severity of periodontal disease, to establish a relationship between the severity of periodontal disease and the species number of representatives of periodontopathogenic microflora.Examination of a patient with periodontal pathology allows not only to correctly diagnose the disease, its severity, and clinical course, but also to determine the etiological factors and pathogenetic mechanisms of the inflammatory or dystrophic process in the periodontium. In this case, it is possible to clarify the role of genetic factors, the influence of nutrition, ecology, occupational hazards, etc. All these examination results form the basis for drawing up an adequate, comprehensive treatment plan using the means of etiotropic, pathogenetic and symptomatic therapy. To predict and plan the complex treatment of periodontal diseases, it is first necessary to identify the etiological factors that predispose to the development of inflammatory changes in periodontal tissues. Achievements in the field of cultivation and study of dental plaque bacteria, identification of differences in the structure of plaques in normal and pathological conditions formed the basis for the search for specific causes of periodontitis . In accordance with the socalled "specific plaque hypothesis", which was formulated in the mid-1970s, the pathogenicity of a plaque depends on the presence or relative predominance of specific microbes in it, and destructive periodontitis is a consequence of the process initiated by them . The adoption of a specific hypothesis was facilitated by the fact that A. actinomycetemcomitans was named as the main causative agent of localized aggressive periodontitis. The concept of biofilms has changed approaches to diseases in various fields of medicine: taking into account new data, the concepts of pathogenesis are being revised . In the development of periodontal diseases, an important role is played by such etiological factors as previous and concomitant diseases, heredity, bad habits (smoking), vitamin deficiency, social factors (stress) . Currently, traumatic occlusion is also referred to as an etiological factor in the development of periodontal diseases. Its appearance is facilitated by factors such as a reduction in the number of pairs of antagonistic teeth, deformation of the dentition, anomalies of individual teeth, dentition and bite, prosthetics with bridges and the use of clasp systems of removable dentures. According to WHO, a high level of periodontal disease is observed at the age of 35 to 44 years (65-98%). It is noteworthy that it is in this age category of patients that factors appear that predispose to the occurrence of recurrent occlusal trauma, which, in turn, provokes the development of periodontal diseases [3, 4] . Since the main etiological factors in the development of periodontal diseases are the microflora of the oral cavity and traumatic occlusion, the analysis of the clinical picture and X-ray examination data allows us to make a differential diagnosis between the forms of traumatic occlusion, justify complex treatment and give recommendations to the patient on caring for the oral cavity after orthopedic treatment . Currently, about 530 species of microorganisms have been identified that live in supragingival and subgingival plaque . A little more than ten are classified as periodontopathogenic, most of them are gram-negative. Obligate anaerobes include Porphyromonas endodontalis, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia. Facultative anaerobes are represented by Aggregatibacter actinomycetemcomitans . The main role in the occurrence of periodontal diseases is played by microorganisms included in the "red complex" BANA +: Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia. With the simultaneous identification of these types, one can judge a high risk of developing periodontal diseases. (BANA is a test for the detection of bacteria containing trepsin-like peptidase, which hydrolyzes the synthetic peptide N-a-Benzoil-DL-arginin-2-naphthylamide (BANA)) . In the periodontal tissues with partial loss of teeth during the chewing function, a functional stress occurs that exceeds the physiological one, which, to a certain extent, will be compensated by the corresponding tissue and vascular reactions of the periodontium. In the future, excessive stretching of the periodontal fibers occurs, which leads to bone resorption and a violation of the position of the tooth. This is the so-called compensated functional overload. With traumatic occlusion, cell damage and the release of lysosomal enzymes, chemical mediators, and collagenase activators occur. Mediators increase vascular permeability, enzymes damage the vascular endothelium, causing tissue exudation and edema, and collagenase destroys fibers . If traumatic occlusion is eliminated, phagocytes absorb the remains of destroyed tissues, fibroblasts again synthesize glycoproteins and collagen fibers, and tissues heal . Purpose -to determine the frequency of occurrence of various types and topography of dentition defects in patients with varying degrees of severity of periodontal disease, to establish a relationship between the severity of periodontal disease and the species number of representatives of periodontopathogenic microflora.
Methodology A clinical examination of a group of patients consisting of 30 women and 8 men in the age category from 35 to 60 years was carried out. The collection of anamnesis was carried out using a survey and filling out a special questionnaire. External examination and examination of the oral cavity made it possible to establish the clinical conditions and anatomical features that contribute to the development of periodontal diseases, namely: the type of bite, the presence of defects in the dentition, traumatic occlusion, plaque, the degree of pathological tooth mobility, bleeding gums, the depth of periodontal pockets. An analysis of 38 orthopantomograms was carried out in order to determine the level of bone tissue destruction in the area of all groups of teeth and establish a final diagnosis. Sterile paper points were used to collect the contents in the area of the deepest periodontal pockets in 4 segments of the jaws. The pins were placed in Eppendorf with a transport medium. In the microbiological laboratory of PIMU, periodontopathogenic microorganisms were identified by PCR diagnostics and their number was measured. The test systems of the Dentoscreen kits make it possible to identify seven microorganisms associated with the development of inflammatory periodontal diseases in a clinically significant concentration. The level of risk of periodontal disease was assessed according to the manufacturer's scale. Results Based on the data of clinical and radiological examinations, patients were divided into three groups depending on the severity of periodontal disease: the first group included 7 patients with a mild degree, the second group included 14 patients with a moderate degree, and the third group included 17 patients with a severe degree. During a clinical examination, we calculated the frequency of occurrence of various kinds of dentition defects in patients with varying degrees of severity of periodontal disease. In patients with a mild degree of periodontitis, included, terminal and combined dentition defects occur with approximately the same frequency, in patients with moderate severity, combined defects are more common, and included and terminal -in 33 and 17% of cases, respectively, in patients with severe severity in 82% of cases, combined defects of the dentition were observed and in 18% -included. After analyzing the results of the PCR study, a direct correlation was established between the severity of periodontal disease and the number of representatives of periodontopathogenic microflora. In the group of patients with mild periodontitis, 65% had up to 2 representatives of pathogenic microorganisms, 35% had more than 4, in the group with moderate severity, 50% had up to 5 microorganisms, and 50% had 6 representatives, in the group with severe degree in 64% -up to 4 microorganisms and in 36%more than 6 representatives. Moreover, the following pattern has been established: the more representatives of the "red complex" BANA + are found in a patient, the more severe the degree of periodontitis. In the group of patients with mild severity of periodontal disease in 89% of cases there are 1-2 representatives of this complex and only in 11% -3, in the group with an average degree in 67% of patients -1-2 representatives and in 33% -3, in in the group with a severe degree in 40% of patients -2 representatives and in 60% -3.
Findings It was found that the degree of generalized periodontitis depends on the number of microorganisms causing periodontal pathology. Given the microbiological status, it is possible to rationally plan orthopedic treatment. [ Development and implementation of an integrative approach to planning and orthopedic treatment of generalized periodontal diseases : avtoreferat diss. cand. med. science].Tver. (In Russ.) 9. Haraeva, Z. F., Mustafaev, M. Sh., Zhanimova, L. R. (2014). Osobennosti mikroflory pacientov s parodontitom raznoj stepeni tyazhesti, vyyavlennye pri pomoshchi PCR metoda [Features of the microflora of patients with periodontitis of varying severity, identified using the PCR method]. Molekulyarnaya diagnostika [Molecular Diagnostics], 1, 258-259. (In Russ.) 10. Bazarnyj, V. V., Polushina, L. G., Maksimova,A.Yu., Svetlakova, E. N., Mandra,Yu. V. (2018). Citologicheskaya harakteristika bukkal'nogo epiteliya pri hronicheskom generalizovannom parodontite [Cytological characteristics of buccal epithelium in chronic generalized periodontitis]. Klinicheskaya laboratornaya diagnostika [Clinical laboratory diagnostics], 12 (63), 773-776. (In Russ.) Microbiology and immunology for dentists]. Moscow 7. Yanushevich, O. O. (2018). Parodontologiya. : Prakticheskaya medicina, 504. (In Russ.) Nacional'noe rukovodstvo [Periodontology. The Na- 4. Mustakimova, R. F., Saleeva, G. T. (2014) tional guidebook]. Moscow : GEOTAR-Media, 752. Komp'yuternaya tomografiya v diagnostike (In Russ.) zabolevanij parodonta [Computed tomography in the 8. Saakyan, M. Yu. (2017). Razrabotka i diagnosis of periodontal diseases]. Zdorov'e cheloveka vnedrenie integrativnogo podhoda k planirovaniyu i v XXI veke [Human health in the XXI century], 192- ortopedicheskomu lecheniyu generalizovannyh 194. (In Russ.) zabolevanij parodonta [ 5. Orekhova, L. Yu., Atrushkevich, V. G., Mihal'chenko, D. V., Gorbacheva, I. A., Lapina, N. V. (2017). Stomatologicheskoe zdorov'e i polimorbid- nost': analiz sovremennyh podhodov k lecheniyu sto- matologicheskih zabolevanij [Dental health and poly- morbidity: an analysis of modern approaches to the treatment of dental diseases]. Parodontologiya [Parodontologiya], 3, 15-17. (In Russ.) 6. Orlova, O. G., Punchenko, O. E. (2014). Vy- yavlenie parodontopatogennyh mikroorganizmov u zdorovyh nositelej s pomoshch'yu molekulyarno-bio- logicheskih metodov [Detection of periodontal patho- genic microorganisms in healthy carriers using molec- ular biological methods] Molekulyarnaya diagnostika [Molecular Diagnostics], 1, 259. (In Russ.)
|
10.1016/j.cub.2018.10.009
|
cc-by
| null | null |
openalex
|
Highlights d C. elegans sleep across most physiological conditions, including developmental arrest d The sleep-active RIS neuron generally induces physiological sleep d Insulin and sirtuin signaling control AMPK and FoxO to induce sleep during starvation d Sleep is required to survive developmental arrest and counteracts aging phenotypes AuthorsINTRODUCTION Sleep supports higher brain functions such as memory consolidation and synaptic plasticity . Sleep disorders are linked to poor health, including the progression of neurodegenerative dis-eases and reduced lifespan in humans. Therefore, the wide prevalence of sleep disorders in modern societies poses a major health problem . Sleep is ancient in origin and most likely evolved together with the emergence of a nervous system. However, little is known about the conditions that led to the evolution of sleep and about how sleep controls basic vital functions. Studying sleep in simple animals can shed light on the essential needs fulfilled by sleep . Sleep is found in all organisms that have a nervous system, ranging from jellyfish to humans . Its widespread occurrence implies that sleep is important, a view supported by the finding that sleep deprivation has detrimental effects . Environmental conditions can impact sleep [6, 7] . Nutrient availability often fluctuates, and all organisms have thus established strategies to sense and respond to a lack of food. Across species, starvation triggers developmental arrest and a biphasic behavioral response consisting of a first phase of increased activity and suppressed sleep, followed by decreased physical activity . Although increased physical activity is understood as a strategy to increase foraging, less is known about the regulation and function of decreased behavioral activity following long-term starvation . Modest food deprivation can exert beneficial effects, suggesting that, in order to survive food deprivation, animals adapt their physiology and activate health-and longevity-promoting pathways [16, 18] . Caenorhabditis elegans is a model animal with low ethical hurdles for harsh survival assays. It lives a boom-and-bust lifestyle with periods of rapid proliferation when food is present alternating with long periods of starvation. As an adaptation to food scarcity, C. elegans has evolved survival strategies, including larval developmental arrest, an alternative larval life stage called ''dauer,'' and an increased lifespan in adults . The study of dietary restriction and starvation and their role on lifespan in C. elegans has led to the identification of major conserved signaling pathways controlling development and aging. Important nutrient-sensing and lifespan-promoting systems act through AMP-activated kinase and the FoxO transcription factor . Like most animals, C. elegans sleeps, a phenomenon that has been studied most in the developing larva and after cellular stress in the adult [22, 23] . From worms to humans, sleep is induced by conserved sleep-active neurons that depolarize at the onset of sleep. They actively induce this behavior by directly inhibiting arousal circuits through GABA and neuropeptides. Upstream pathways control the depolarization of sleep-active neurons and thereby control the timing and amount of sleep [24, 25] . In mammals, several populations of sleep-active neurons exist, which are thought to induce sleep in a concerted action. The best-studied population of sleep-active neurons is found in the preoptic area (POA). These neurons form part of the so-called descending system that inhibits arousal during sleep. Sleep-active neurons also confer an increased sleep pressure after sleep deprivation, as their depolarization is increased at the onset of rebound sleep [26, 27] . Similarly, several populations of sleep-promoting neurons exist in Drosophila. A cluster of nerve cells innervating the dorsal-fanshaped body (dFB) of the central complex presents a well-studied population of sleep-promoting neurons, whose excitability can be switched dependent on sleep need . In C. elegans, sleep during lethargus, a developmentally controlled phase of molting during which worms synthesize a new cuticle, requires a single sleep-active sleep-inducing neuron called RIS, which expresses GABA and inhibitory RFamide peptides. Like its mammalian counterparts, this neuron induces sleep when depolarized optogenetically, shows calcium transients at sleep onset, most likely indicating depolarization, shows over-activation after sleep deprivation, and is inhibited by waking stimuli . To find out why a simple animal sleeps, we searched for conditions in which RIS-induced sleep is essential for survival in C. elegans. Although worms show sleep during most stages and conditions, this behavior is most prominent during various forms of starvation and developmental arrest, where conserved nutrient-sensing lifespan regulators control it. Although sleepless adult worms have a normal lifespan under both ample food condition as well as starvation, sleeplessness impairs survival of larvae during developmental arrest. Interestingly, the role of sleep in starvation-induced arrest survival appears not to only be conservation of energy but to prevent the progression of aging phenotypes. Thus, sleep presents an adaptive anti-aging strategy to survive starvation-induced arrest. Our work provides a molecular link between sleep, longevity, starvation, and developmental arrest with high potential implications for the evolutionary origin of sleep as well as for human health.
RESULTS
Sleep Is Widespread across Stages and Conditions, with Extended Starvation Presenting a Major Sleep Trigger To find out how sleep becomes vital in a simple animal, we quantified sleep during several life stages and conditions to find out what is the strongest trigger for this state. We focused on food availability and quality, which affects behavioral activity and quiescence [10, 11, 19, 32, 33] . We used RIS calcium imaging as a proxy for depolarization of this neuron and locomotion quiescence as an assay to identify and quantify sleep. Worms were cultured in microfluidic devices made from hydrogel [34, 35] and behavior, and calcium transients in RIS were imaged and quantified during different life stages and food conditions (Figures 1 and S1 ). We first looked for sleep in adult worms and tested the effects of three types of bacterial food: first, to mimic standard worm culture conditions, we fed worms with bacteria in the presence of bacterial growth medium (Figure 1A ). Second, we tested bacteria that were depleted of growth medium (Figure 1B ). And third, we tested dead bacteria in the presence of growth medium (Figure 1C ). Worms living on fed bacteria showed extended sleep bouts (Figures 2A, S2A , and S2B). A reduction of locomotion always coincided with increased RIS depolarization (Figure 1J ). Worms feeding on dead or starved bacteria, however, showed virtually no sleep behavior (Figures 1B, 1C, and 2A ). Short-term (few hours) fasting results in increased arousal and foraging in C. elegans , whereas prolonged (more than 12 hr) starvation results in behavioral quiescence and developmental arrest [10, 11, 19, 20, 33, 36] . To analyze sleep during starvation and arrest, we looked at one day starved adults, L1 larvae during lethargus (when worms do not feed during cuticle remodeling), one day starved developmentally arrested L1 larvae, and three days starved dauer larvae. We observed prominent sleep bouts during starvation and arrest in all stages that were tested (Figures 1D-1H, 2B-2E, and S2C-S2J). RIS again depolarized strongly at the onset of all sleep bouts (Figures 1K-1N ). As pheromones play a strong role in anticipation of adverse life conditions and the development of the developmentally arrested dauer larva , we also tested the effect of pheromones on sleep in adults feeding on starved bacteria. Dauer pheromone (A) An adult worm feeding on OP50 bacteria with bacterial growth medium. (B) An adult worm feeding on OP50 without bacterial growth medium. (C) An adult worm feeding on dead OP50 with growth medium. (D) An adult that was starved for 24 hr. (E) An L1 larva in the presence of food, 8 hr after hatching and prior to lethargus. (F) An L1 larva before and during lethargus as defined as the non-feeding period (starting at 0 hr) prior to the molt in the presence of food. (G) An arrested L1 larva that was starved for 24 hr. (H) A 3-day-old dauer larva in the absence of food. (I) An adult feeding on OP50 without growth medium in the presence of dauer pheromone. (J-O) RIS activity increased significantly with locomotion cessation at the onset of sleep bouts in all conditions. The increase of D F/F of the calcium sensor signal was (J) 126.1% ± 20.2%, n = 24 worms, *p < 0.001 for adults on growing OP50; (K) 32.5% ± 5.4%, n = 20 worms, p < 0.01 for starving adults; and (L) 22.0% ± 0.9%, n = 14 worms, *p = 0.014 for developing L1 worms during lethargus. (M-O) 58.7% ± 4.9% (M), n = 33 worms, *p < 0.001 for arrested L1 larvae; 36.8% ± 7.9% (N), n = 13 worms, p = 0.002 for dauer larvae; 45.7% ± 19.8% (O), n = 12 worms, *p = 0.02 for adults on stationary OP50 with dauer pheromone. For all statistical comparisons, the paired Wilcoxon rank test was used. See also Figures S1 and S2 . extract also induced sleep bouts and RIS depolarization, which is consistent with the observation that population density affects sleep (Figures 1I, 1O, 2F, S2K, and S2L) . In summary, behavioral quiescence was typically accompanied by RIS activation at the onset of virtually all sleep bouts and across all stages and conditions that we tested. An increase in RIS calcium precisely correlates with reduction of behavioral activity. Thus, rather than reflecting sleep pressure building up during wakefulness prior to sleep onset [11, 38] , RIS activity indicates the active induction of sleep. Food conditions appear to be a major determinant for RIS activation and sleep amounts in both larvae and adults. Not only the presence or absence of food determines sleep quantity but also food quality, with metabolizing bacteria appearing as a strong sleep trigger. Extended starvation and arrested development triggered the strongest sleeping behavior. As pheromones signal population density, sleep may already be modulated in anticipation of starvation. RIS calcium imaging combined with locomotion quantification thus presents a straightforward assay to identify sleep, which occurred during all stages and most conditions and thus is much wider spread than previously thought [22, 39] . Fraction of time spent asleep in wild-type, aptf-1(gk794), and RIS-ablation (RIS(À)). (A) Median sleep duration in adult worms in the presence of growing bacteria was 18% in wild-type and 8% in aptf-1(gk794); *p < 0.001. In the absence of growth medium and in the presence of dead bacteria, median sleep duration was 0% for both wild-type and aptf-1(gk794 [10, 11, 19, 32, 33, . Sleep is defined and can be distinguished from other types of quiescence by a reduced response threshold, rapid reversibility, and homeostatic regulation . We hence assayed whether quiescence during L1 arrest fulfills the behavioral definition of sleep. To test for responsiveness, we used blue light to trigger an escape response in arrested L1 larvae inside micro-fluidic compartments and measured the resulting increase in locomotion. Although waking worms responded with a strong locomotion increase, during sleep, the acceleration was reduced (Figures 3A and 3B ). We next tested for reversibility of sleep by providing a strong optogenetic sensory stimulus. For this, we expressed the channelrhodopsin variant ReaChR in nociceptive ASH neurons or mechano-sensory neurons, depolarized them with green light , and followed locomotion and RIS polarization. Optogenetic stimulation of ASH neurons or mec-4-expressing neurons quickly reversed quiescence (Figures 3C, 3D, S3A, and S3B ). The waking stimulus also acutely inhibited RIS activity, leading to a premature termination of the RIS depolarization transient (Figures 3C, 3D , S3A, and S3B). To provide a non-optogenetic sensory stimulus, we applied noxious blue light, which triggers an avoidance response [45, 47] . Similar to the optogenetic stimulus, blue light reversed quiescence and inhibited RIS (Figures 3E and S3C ). To test for homeostatic regulation, we deprived sleep through temporary optogenetic inhibition of RIS using the light-driven proton pump ArchT . We inhibited RIS for 1 hr using green light and measured the behavioral response and the depolarization of the RIS neuron before, during, and after the inhibition. Optogenetic silencing efficiently prevented RIS depolarization and sleep induction during illumination (Figures S3D and S3E ). Following the end of RIS inhibition, sleeping behavior and RIS depolarization increased compared to control levels, suggesting that quiescence is under homeostatic control (Figures 3F, S3D, and S3E ). During sleep, including C. elegans lethargus, the majority of neurons show dampened activity [38, 49] . To test whether global neural activity is reduced during sleep, we imaged pan-neuronal calcium activity. Global brain activity was indeed dampened during quiescence bouts (Figure 3G ). Thus, starvation-induced quiescence during larval arrest presents a sleep state, with its key behavioral and neurophysiological hallmarks. Regulatory principles typical for mammalian sleep are present also in starvation-induced sleep: the fast inhibition of RIS through a waking stimulus is similar to the flip flop switch that ensures discrete behavioral states and the increased RIS (C) Sleeping worms responded immediately to ASH activation and the sleep bout ceased. The fraction of sleeping animals decreased by 91.6% ± 1.7% (control worms without ATR by 12.3% ± 2.9%; *p < 0.001; two-sample t test). RIS activity (DF/F) dropped by 35.5% ± 4.2% (control worms without ATR showed only a drop of DF/F of 11.9% ± 0.3%; *p < 0.001 two-sample t test). Locomotion velocity increased by 313.7% ± 26.0% (control worms without ATR by 65.7% ± 14.0%; *p < 0.001; two-sample t test). n (ATR) = 23 worms; n (without ATR) = 47 worms. (D) Sleeping worms immediately responded to mec-4-expressing mechano-sensory neuron activation, and the sleep bout ceased. The fraction of sleeping animals decreased by 52.45% ± 6.0% (control worms without ATR by 10.0% ± 2.6%; *p < 0.001; two-sample t test). RIS activity (DF/F) dropped by 32.8% ± 3.2% (control worms without ATR showed only a drop of DF/F of 7.0% ± 2.1%; *p < 0.001; two-sample t test). Locomotion velocity increased by 351.7% ± 44.0% (control worms without ATR by 14.6% ± 4.0%; *p < 0.001; two-sample t test). n (ATR) = 29 worms; n (without ATR) = 50 worms. (E) Sleeping worms immediately responded to noxious blue light illumination, leading to sleep bout termination. The fraction of sleeping animals decreased by 75.5% ± 3.5% (control worms without blue light stimulation by 4.1% ± 0.6%; *p < 0.001; two-sample t test). RIS activity (DF/F) dropped by 43.2% ± 7.9% (control worms without blue light stimulation showed only a drop of DF/F of 4.3% ± 7.7%; *p < 0.001; two-sample t test). Locomotion velocity increased by 367.9% ± 5.8% (control worms without ATR by 80.3% ± 28.2%; *p < 0.001; two-sample t test). n (stimulation) = 35 worms; n (without stimulation) = 37 worms. (F) To test for homeostasis, RIS was inhibited for 1 hr with ArchT and green light in the arrested L1 larvae. RIS activity is shown in red (control without ATR in light red), speed in black (control without ATR in gray), and the fraction of sleeping animals in blue (control without ATR in light blue). After the end of green light exposure, the fraction of sleeping worms increased by 265.6% ± 13.1% (control worms without ATR decreased by 19.3% ± 6.7%; p = 0.007; two-sample t test). RIS activity (DF/F) increased by 24.3% ± 2.2% (in control worms without ATR, it increased by 5.6% ± 1.0%; p = 0.005; two-sample t test). Locomotion velocity decreased by 63.3% ± 16.0% (control worms without ATR increased by 5.5% ± 4.0%; *p < 0.001; two-sample t test). n (ATR) = 32 worms; n (without ATR) = 25 worms. (G) To test for overall neuronal activity during a sleep bout, calcium activity in the head neurons was measured using pan-neuronal GCaMP6s. Head neuron activity is shown in orange and locomotion speed in black; blue shading shows sleep bouts as defined by a locomotion cessation threshold. At the onset of a sleep bout, head neuron activity decreased and locomotion ceased. depolarization during rebound sleep also occurs similarly in sleep-active neurons of the vertebrate POA .
AMP-Activated Kinase and FoxO Act in Parallel to Induce Sleep during Starvation Starvation is sensed by conserved pathways that trigger protective strategies. AMP-activated kinase (AAK-1/2) is an energy sensor that is activated by a high AMP/ATP ratio and that triggers the switch from anabolic to catabolic processes. Reduced activity of the insulin/insulin-like signaling (IIS) receptor (DAF-2) leads to the activation of FoxO (DAF-16), a transcription factor that expresses genes that promote stress resistance and longevity . DAF-16 also plays a role in coping with the consequences of stressful sleep deprivation and modulates quiescence [11, . Both FoxO and AMPK are required for starvationinduced developmental arrest [20, 53] . To test whether longevity pathways are responsible for the majority of starvation sleep, we measured RIS activation and sleep in mutants that are defective in AMP-activated kinase and IIS. We first tested a loss-of-function mutation in daf-2 in the presence of food conditions that normally do not lead to sleep. daf-2(À) caused sleep and RIS depolarization in the presence of starved bacteria as a food source (Figures 4A-4C, S2M, and S2N), and this effect was dependent on daf-16 (Figure 4C ). We next tested sleep in daf-16(À) during larval and adult starvation. daf-16(À) showed only a moderate reduction of sleep in adults, consistent with previous results (Figure 4D ) . Similarly, partially reduced sleeping behavior was found in aak-1/aak-2 double-mutant animals during starvation (Figure 4D ), prompting us to knock out both pathways simultaneously. The aak-2/daf-16 double mutant showed a near-complete absence of sleep in the adult (Figure 4D ), but not in the larva (Figure 4E ). aak-1/aak-2/daf-16 triple mutant worms showed a strong loss of sleep in the starved adult (Figure 4D ) and also a strong sleep reduction in the arrested L1 larva (Figure 4E ). Thus, AAK-1/2 and DAF-16 appear to act in parallel to induce sleep during extended starvation. We next wished to link starvation-induced sleep to the genes that are upstream of AMPK and FoxO. We hence quantified sleep in mutants of genes that are known to control AMPK and FoxO. In the presence of food, IIS activates the phosphatidylinositol 3-kinase (PI3K) AGE-1 to produce PIP3, which activates Akt kinase (AKT-1/2) . A major role of Akt in C. elegans is to inhibit FoxO . In other systems, Akt also inhibits AMPK by adding an inhibitory phosphorylation on Ser485, which then inhibits the activation of AMPK by LKB1 , and there is evidence that Akt also inhibits AMPK in C. elegans . Thus, both FoxO and AMPK have in common that they can be suppressed through Akt . The tumor suppressor DAF-18/PTEN inhibits IIS signaling by hydrolyzing PIP3 produced by AGE-1/PI3K. daf-18 deletion thus leads to constitutive activation of Akt . We tested whether daf-18 deletion leads to a suppression of starvation-induced sleep and found that sleep was strongly reduced (Figure 4F ). Starvation also leads to the activation of SIRT/ SIR-2.1, which can act through activation of both FoxO and AMPK . We thus tested sleep after deletion of sir-2.1. We observed again a reduction of sleep in sir-2.1(À) mutants (Figure 4F ). These results are consistent with the known network of longevity genes and a model in which, in the presence of food, canonical IIS is wake promoting through the inhibition of AMPK and FOXO and in which, during starvation, sirtuin signaling is sleep-promoting by activating AMPK and FOXO (Figure 4G ). Such dual control mechanism of two parallel pathways could account for the robustness of sleep induction during starvation. Our data thus suggest that the known network of longevity genes controls sleep induction during starvation. To find out in which tissues AMPK and FoxO act during starvation to induce sleep, we performed rescue experiments. We used daf-2(À)/daf-16(À) worms and expressed daf-16 specifically in either neurons, muscle, or intestine and quantified sleep in adults in the presence of food [59, 60] . Sleep was reinstated partially but significantly by expression in muscle and inconsistently by expression in intestine, but not by expression in neurons (Figure S4A ). The role of daf-16 in muscle during starvation is consistent with previous data suggesting a role of daf-16 in muscle to control increased sleep after forced locomotion during lethargus and supports a conserved role of muscle in signaling sleep need [51, 61] . During physical exercise, musculature is the major consumer of energy, and this tissue may thus be well suited to signal energy shortage during both forced movement and starvation . The weak rescue from non-neuronal tissues together with the lack of neuronal rescue suggests that daf-16 acts in signaling centers outside of RIS. To identify the tissues in which AMPK acts, we expressed aak-2 in an aak-2(À)/daf-16(À) background. We quantified sleep in adult worms in the absence of food and looked for rescue in neurons, muscle, intestine, hypodermis, and the excretory cell [15, 56, 63] . AMPK rescue effects were partial but significant in all tissues that were tested. Because AMPK could be rescued from all tissues, including neurons, we specifically tested whether AMPK also can act in RIS and found that it partially could (Figures S4B and S4C ). Thus, AMPK appears to be able to induce sleep by acting in many tissues, including in RIS. Consistent with previous reports, which found that AMPK can act across various tissues, this may indicate that many or perhaps even all tissues that experience energy stress can signal to RIS for sleep induction [15, 56, 63] .
Sleep Is Required to Survive Larval Starvation-Induced Arrest The strong induction of sleep during starvation and arrest through aging pathways suggested that sleep might play a role in surviving food deprivation and development cessation [18, 64] . Thus, we tested adult lifespan along with survival and recovery from L1 arrest in the presence or absence of food in sleepless mutants. We first measured the lifespan of adult worms in the presence of food and during starvation. In both conditions, there was no consistent and significant difference (G) Hypothetical working model that integrates literature data with our observations. The numbers of assayed worms (n) are displayed below the boxplots. Sleep duration comparisons with N2 were made using the Mann-Whitney U test and were confirmed with Benjamini-Hochberg procedure for multiple comparisons. Error bars indicate the SEM. See also Figure S4 in lifespan between wild-type, aptf-1(À), and RIS-ablated worms (Figures 5A-5D and S5A-S5D). We then tested L1 arrest survival and recoverability. For this, we measured both the time that L1 larvae stayed alive in the absence of food as well as the time until larvae were still able to recover from the arrest and to resume their development when fed . Both survival time and the ability to recover were reduced consistently and significantly by about half in sleepless mutants (Figures 5E. 5F, and S5E-S5H). Thus, although there does not seem to be a strict requirement of sleep for survival in adult C. elegans, it becomes essential in the arrested larva. To test whether sleep is a predictor of survival, we quantified quiescence for individual starved L1 larvae inside microfluidic devices and also measured their survival span. A correlation was seen between the amount of sleep during the first 2-4 days of starvation and survival (an increase of 1% sleeping time corresponded to an increase in survival extension by roughly 5%; Figure 5G ), suggesting that sleep is a predictor for surviving starvation. If sleep is part of the protective program induced by AAK-1/2 and IIS, then ablating sleep in AAK-1/2 and IIS mutants should not further decrease survival during larval starvation. To test this idea, we looked at survival of aptf-1(À) mutant worms in a FoxO/AAK-1/2 triple mutant background. There was no difference in survival due to sleep loss when we compared the daf-16(mgDf50)/aak-1(tm1944)/aak-2(ok524) triple mutant with daf-16(mgDf50)/aak-1(tm1944)/aak-2(ok524)/aptf-1(gk794) quadruple mutant (Figures 5H and S5I ). This is consistent with the view that sleep and AAK/FoxO act in the same pathway. If the role of sleep were to conserve energy or nutrients, then feeding would suppress the requirement of sleep for survival. To test this idea, we arrested L1 larvae using 5-fluoro-2 0 -deoxyuridine (FUdR), kept them in the presence of ad libitum E. coli as food source, and measured survival. The arrested larvae consumed the bacteria (Figures S6A and S6B ) and showed sleeping behavior (Figure S6C ). However, sleepless worms still had a significantly shorter lifetime than the sleeping control (Figures 5I and S5J ). This suggests that, although resource preservation is important, sleep acts beyond energy or nutrient conservation to achieve survival.
Sleep Counteracts the Progression of Aging Phenotypes during Starvation-Induced Arrest Recent work demonstrated that L1 arrested larvae show a progression of aging phenotypes leading to death. Thus, the cause of death of arrested larvae is similar to the one in the adult . We wondered whether sleep is required for survival by slowing down the progression of aging phenotypes during starvation. To test this idea, we looked at established markers of aging, such as the deterioration of body wall muscle fibers, mitochondrial fragmentation, and protein aggregation . We crossed aptf-1(À) into transgenic strains in which either muscle myosin, muscle mitochondria, or an aggregation-prone polyglutaminecontaining protein, poly-Q35::YFP, were fluorescently labeled . L1 larvae were starved, and the progression of aging was monitored by fluorescence microscopy until the animals had died. The morphology of the aging markers looked normal in freshly starved L1 aptf-1(À). However, as time passed, aging markers progressed more quickly in the sleepless mutant compared with the wild-type. Muscle fibers degraded more quickly, mitochondria underwent fission earlier, and protein aggregation occurred faster. Sleepless worms reached the same maximal level of aging marker progression approximately ten days earlier compared with the wild-type (Figure 6 ). Together, these results suggest that, rather than merely saving energy, sleep is required for survival of starvation by counteracting the progression of aging processes.
DISCUSSION To understand why sleep is important for C. elegans, we monitored behavioral activity and RIS calcium transients to survey the prevalence of sleep across life stages and physiological conditions. We tested for the effects of food quality and availability, as these define the major physiological conditions in the life of C. elegans, which proliferates in ephemeral microbe-rich habitats and is able to survive long periods of starvation . Sleep occurred during most stages and conditions that were analyzed. RIS depolarized at the onset of sleep bouts and typically showed activity until the end of the sleep bout, inversely mirroring the pattern of behavioral activity. This pattern of RIS depolarization is consistent with the view that RIS is the inducer of sleep, and the maintenance of high RIS activity throughout most of the sleep bout suggests that sleep is under constant control of RIS. During longer sleep bouts, which were, for example, observed in dauer larvae and daf-2(À), calcium transients still correlated with sleep onset but decreased already before the end of the bout. This may speculatively be caused by an additional mechanism that may dampen wakefulness independently of RIS, thus potentially explaining a reduced requirement for RIS activity for maintaining sleep. Consistent with this speculation, spontaneous motion is reduced in daf-2(À) and dauer larvae . daf-2(À) mutants showed a residual fraction of immobility that could not be suppressed by aptf-1(À). Similarly, a small fraction of residual quiescence was observed in the absence of functional RIS when worms were fed with bacteria in the presence of growth medium. Different scenarios could explain this residual quiescence. First, occasionally, slow locomotion may be falsely scored as quiescence by the analysis algorithm. Second, quiescence may exist that is not sleep or is sleep but is not induced by RIS. Severe cellular stress can also induce behavioral quiescence independently of RIS, involving activation of the ALA neuron [39, 41] . Thus, hypothetically, some of the conditions that we (H) Starved arrested L1 aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50)/aptf-1(gk794) quadruple mutants showed no reduced survival compared to aak-1(tm1944)/ aak-2(ok524)/daf-16(mgDf50) triple mutants (time at which 50% of worms were still alive, aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50): 3.9 days, n > 60 worms; aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50)/aptf-1(gk794): 3.9 days, n > 60 worms; p > 0.05 for all days; Fischer's exact test). (I) FUdR-arrested L1s in the presence of food have a decreased survival. Arrested L1 survival is significantly decreased in aptf-1(gk794) and in RIS(À) worms (mean survival, N2: 25.7 days, n = 143 worms; aptf-1(gk794): 21.9 days, n = 140 worms; *p < 0.001; RIS(À): 22.1 days, n = 135 worms; *p < 0.001; log rank test). See also Figures S5 and S6 and Tables S1, S2, S3, S4, and S5 for additional replicates and details on food consumption. investigated may have also triggered a stress response and RIS-independent quiescence. Third, in some instances, the genetic RIS ablation might not have been fully penetrant due to transgene silencing or mutant suppression. Because, overall, only a minority of quiescent counts did not depend on RIS, we did not further investigate their cause. In summary, it emerges that, under physiological conditions, the majority of behavioral quiescence in C. elegans presents sleep caused by RIS. In C. elegans, sleep amount strongly depends on food quality and availability, with starvation, developmental arrest, and diapause presenting the strongest sleep triggers. We hence focused our analysis on the role of sleep during starvation conditions. Starvation and aging are intimately linked: seminal work from C. elegans has led to the identification of genes controlling aging, including FoxO and AMP kinase, and these healthy aging genes are also required for surviving starvation [20, 21] . Genetic analysis in mice and humans showed that these genes are conserved regulators of aging . Here, building on previous work on aging and sleep in C. elegans [11, 36, 51, 52] , we linked the network of known longevity genes to sleep induction. This longevity network consists of IIS and sirtuin signaling, which control FoxO and AMPK, which activate the RIS neuron and thus induce sleep. FoxO and AMPK can act in tissues outside of RIS (even though AMPK can also act within RIS), suggesting that multiple tissues can signal starvation to induce sleep. All organisms are threatened with the possibility of experienced famine in their life. Thus, strategies to survive the depletion of food are highly adaptive and underlying mechanisms are deeply conserved. Across species, animals show a stereotyped biphasic behavioral response to starvation. First, arousal is increased and sleep is reduced . This phase is then followed by a phase of reduced physical activity . Extreme conditions of food deprivation can lead to a quiescence state known as torpor, which is thought to preserve energy by shutting down physical and metabolic activity. Torpor differs from sleep in that the behavioral quiescence is not readily reversible. By contrast, sleep has been proposed to not only conserve energy but also to allocate resources [17, 71] . Does sleep serve different or similar functions during different conditions? What are the identities of these functions? And how are they carried out? Sleep has been proposed to serve diverse roles, ranging from energy conservation and allocation [6, 71] and restoration and regulation of key cellular and metabolic processes [72, 73] to higher brain functions [74, 75] . Viability assays revealed an important and specific role of sleep in surviving starvation-induced developmental arrest. Although sleep is vital for surviving starvation in larvae, we did not find any evidence that it is essential for lifespan extension in adults. Consistent with this finding, juvenile stages typically sleep more and are more susceptible to sleep deprivation also in other species [23, 76, 77] . A major biological difference between starvation in adult and larval worms is that adult worms have already completed development, with all somatic cells being postmitotic, whereas the larva has to arrest their development, which includes the inhibition of cell division . Thus, the survival experiments suggest that the vulnerability of larvae to sleep loss relates to the arrested development. Because adult worms also spent substantial time asleep, this suggests that sleep may also serve additional functions, which may not be essential for survival under ideal laboratory conditions [50, 51] . How does sleep prevent death during starvation-induced arrest? The progression of aging phenotypes, but not residual energy reserves, has been shown to predict the ability of larvae to survive and recover from starvation-induced arrest, suggesting that arrested worms do not die because they run out of energy but because of cellular demise that is mechanistically similar to aging . Consistent with this finding, we observed that sleep slows down the aging phenotype progression during starvationinduced arrest. Thus, sleep appears to be required for starvation survival, not only because it saves energy from being burned for motility but rather because it counteracts aging progression. Speculatively, sleep might allocate resources away from behavioral activity toward cell-protecting pathways that are similar to anti-aging processes. Sleep is ancient and most likely evolved to serve basic, essential needs, such as recuperation from stress and illness [41, 79] . Here, we show that sleep during starvation-induced developmental arrest presents an anti-aging program required for survival. This function could have been an important ancient reason for a sleep response, which was co-opted later during evolution to also serve higher brain functions. Most likely, the molecular link between starvation, development, aging, and sleep also plays a role in human sleep. Figuring out how sleep protects against cellular demise will be instructive to understand its revitalizing functions. (C and D) Mitochondrial fragmentation is increased in aptf-1(À). (C) Mitochondrial morphology appears normal in one day starved larvae, but the initially tubular mitochondria fragmented much faster in aptf-1(À) compared with the wild-type. Mitochondria in body wall muscles were labeled using GFP and were classified as tubular (M [tub.]), intermediately fragmented (M [int.]), or completely fragmented (M. [frag.]). Shown are the mid-body regions of worms, and arrows point to example mitochondrial structures or nucleus (N). (D) For the quantification of mitochondrial deterioration, the percentage of muscle cells displaying each level of fragmentation was determined (12 days-M (frag.): n > 20, N2: 8.5% ± 3.3%; aptf-1(gk794): 47.9% ± 5.9%, *p < 0.001; 14 days-M (frag.): n > 20, N2: 15% ± 3.4%; aptf-1(gk794): 56.1% ± 6.1%, *p < 0.001, two-sample t test). (E and F) Protein aggregation is increased in aptf-1(À). (E) Poly-Q35 was expressed in muscle, and the number of aggregates formed was scored. In one day starved L1, there were almost no detectable aggregates in both wild-type and aptf-1(gk794). Shown are the mid-body regions of worms with arrowheads pointing at aggregates. (F) The number of aggregates increased much faster in aptf-1(gk794) compared with wild-type (12 days: n > 20, N2: 1 ± 0.2; aptf-1(gk794): 3.4 ± 0.4, *p < 0.001; 14 days: n > 20, N2: 2.5 ± 0.4; aptf-1(gk794): 4.7 ± 0.7, p < 0.01, two-sample t test).
STAR+METHODS KEY RESOURCES
CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Henrik Bringmann (Henrik.Bringmann@mpibpc.mpg.de).
EXPERIMENTAL MODEL AND SUBJECT DETAILS Worm maintenance and strains C. elegans was grown on Nematode Growth Medium (NGM) agarose plates seeded with E. coli OP50 and were kept at 20 C . For crossings, the strains were genotyped using Duplex PCR genotyping of single worms . Primer sequences used for Duplex PCR can be found in Table S7 . Following C. elegans strains were used: the unc phenotype as a selection marker [91, 92] . The insertions that were obtained were backcrossed two times against N2 to remove the unc-119(ed3) background. The following plasmids were created for this study:
Dauer pheromone extraction and dauer larvae generation The crude dauer pheromone was extracted as described previously . Briefly, worms were cultured in 1L of S-medium with resuspended OP50 at 25 C. When the culture clarified as a consequence of bacteria depletion, additional OP50 was added. When cleared the second time, the supernatant was filtered and boiled to a solid crust. This crust was extracted three times with ethanol. After evaporation of the ethanol, the dauer pheromone was resuspended in 1 mL of sterile water and stored at À20 C. A 10 mL drop of water containing dauer pheromone was placed on the solidified agarose, which contained the microchambers. The drop was placed after sealing the chambers with a coverslip. The drop was pipetted onto the surface that did not contain any microchambers and subsequently diffused into the agar. To obtain dauer larvae, NGM plates were left to starve at 25 C and daily controlled for occurrence of dauer larvae. 3 days after the first dauers occurred, the starved NGM plate was chunked to a fresh unseeded NGM plate and the dauers were allowed to crawl off that chunk. From there, individual dauer larvae were isolated and used for behavioral experiments
Long-term imaging For long-term imaging, agarose microchamber imaging was used [34, 35] . Briefly, box-shaped indentations in agarose hydrogel were cast using a PDMS mold. The chambers were then filled with worms and for fed conditions with bacteria, and sealed with a glass coverslip. 3% agarose dissolved in S-Basal was used as a hydrogel in all experiments to starve worms and bacteria except for the experiments were we investigated behavior in the presence of growth medium. To obtain growth medium conditions we mixed Nematode Growth Medium and S-Basal 1:1, added 3% agarose, and casted microchambers from this mix. To obtain dead bacteria, the OP50 lawn was pasteurized on seeded NGM plates for 3 hr in an oven set to 70 C. For pheromone experiments, 10 mL of dauer pheromone extract was added on top of the agarose microchamber, which then diffused into the agarose. Behavioral and functional calcium imaging [29, 86] was performed simultaneously. In short, we used an Andor iXon (512x512 pixels) EMCCD camera and LED illumination (CoolLed) using standard GFP filter sets. The exposure time was set to 5ms. The EMCCD ''TTL fire out'' signal was used to trigger the LED illumination in order to illuminate only during exposure. The 490nm light intensity was 2.00mW/mm 2 using a 20x objective and 0.60mW/mm 2 using a 10x objective. EM gain was set to 100. Using these parameters, we obtained image sequences with clearly identifiable worm outlines and measurable neuronal calcium transients. Typically, 8-12 individual fields in close vicinity were filmed. Adult worms were cultured in 700mm x 700mm x 45mm (X length x Y length x Z depth) microchambers. Dauer larvae were kept in 370mm x 370mm x 25mm microchambers and L1 larvae in 190mm x 190mm x 15mm unless otherwise mentioned. For adults and dauer larvae, one imaged field included an individual worm in a single microchamber. For L1s, one filmed field can include up to four worms in adjacent microchambers. Adults were imaged using the 10x objective. L1s and dauer were imaged with the 20x objective. An automatic stage (Prior Proscan 2 or 3) moved repeatedly to the microchambers using low acceleration speeds. The frame rate we obtained was 0.1frame/s unless otherwise noted. Worms were filmed either continuously or in series of movies for optogenetic experiments. In the L1 experiments, pretzel stage eggs were picked with an eyelash or eyebrow hair to an empty NGM plate to remove remaining bacteria. From this empty NGM plate, the eggs were transferred to the microchamber again using a clean hair. Image acquisition was started 24 hr after hatching. Dauers were obtained from plates that were left to starve at 25 C. To isolate individual dauers, we cut out agarose chunks of the starved plates and transferred them to an empty NGM plate and allowed the worms to crawl off the agarose chunk. Individual dauers were pipetted with 1-5mL of water to the microchamber. With a pick, the dauers were distributed over the microchambers so that each dauer was placed into its own chamber. We assayed dauers that were 3 days old. Adults for starvation, daf-2(e1370) mutants and its controls were treated with 5-fluoro-2-deoxyuridine (FUdR, Sigma-Aldrich) before the experiments. For this, L4s were transferred to seeded NGM plates containing 50mM FUdR and 50mg/L kanamycin (Sigma-Aldrich) . 24 hr after the FUdR treatment, we transferred the adults to the microchamber. The worms were either transferred with a worm pick into a microchamber filled with OP50 for feeding experiments or they were pipetted in a 1-5mL drop of water to a microchamber for starvation experiments. For starvation experiments, worms were kept without food for 24 hr in the empty microchambers before image acquisition. For daf-2(e1370) mutant experiments, imaging was started 35 hr after shifting the temperature from 15 C to 23 C. Imaging was started 6 hr after the pheromone application. Imaging was started 3 hr after the microchamber was prepared. To allow identification of the non-pumping period, additional DIC images were collected.
Optogenetics and light stimulation All optogenetic experiments using ReaChr and ArchT were performed inside microchambers . Two days before the experiments, the strains were grown on NGM plates supplemented with 0.2mM all-trans retinal (ATR, Sigma-Aldrich). The agarose that was used for microchamber fabrication was also supplemented with ATR of the same concentration. A dual GFP/mCherry excitation/dichroic plus GFP emission filter set (Chroma) was used to allow simultaneous GCaMP imaging and optogenetic manipulation. To activate ReaChr and ArchT, the LED light intensity at 585nm was set to 0.47mW/mm 2 using a 20x-objective and 0.14mW/mm 2 using a 10x-objective. The light intensities were measured using a light voltmeter (PM100A, Thorlabs). To test for responsiveness to stimulation, we applied blue light irradiation at 490nm with an intensity of 3mW/mm 2 to arrested L1 larvae inside the microfluidic compartments. To evoke a behavioral response we used blue light, which triggers an endogenous lightavoidance response. For these experiments, L1 worms were starved for 24 hr inside microchambers (110mm x 110mm x 10mm) and imaged with 2frames/s. After 1min of baseline imaging, the animals were illuminated for 3min and followed by an additional 1min imaging without illumination. Each animal was assayed up to 16 times with at least 1 hr between two blue light irradiations to allow the worm to recover from the stimulus. To extract sleep-wake-differences of responsiveness, the worm's behavioral state was classified post hoc into either ''wake'' or ''sleep.'' The worm was classified as ''wake,'' when it was never in a sleep bout during the imaging sequence 3.5 min before the stimulation (see below) throughout the baseline measurement and was classified as ''sleep'' when it was continuously in a sleep bout during the 3.5 min before stimulation. Only data from time points that continuously met the criteria for being asleep or awake during the 3.5 min before stimulation were taken for the sleep and wake analysis, the other data were excluded. These excluded data were those time points during which the worm showed episodes of both sleep and wake during the 3.5 min of stimulation. The specific numbers of experiments and data exclusions are specified as follows: For Figure 3A , a total of 600 measurements were made. 446 (74.3%) were counted as ''wake,'' 98 (16.3%) were counted as ''sleep'' and 56 (9.3%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3C with ATR, a total of 125 measurements were made. 70 (56.0%) were counted as ''wake,'' 19 (15.2%) were counted as ''sleep'' and 36 (28.8%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3C without ATR, a total of 273 measurements were made. 110 (40.3%) were scored as ''wake,'' 40 (14.7%) were scored as ''sleep'' and 123 (40.1%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3D with ATR, a total of 167 measurements were made. 101 (60.5%) were counted as ''wake,'' 42 (25.1%) were counted as ''sleep'' and 24 (14.4%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3D without ATR, a total of 297 measurements were made. 69 (23.2%) were counted as ''wake,'' 85 (28.6%) were counted as ''sleep'' and 143 (48.1%) could not be assigned to ''wake'' or ''sleep'' and were excluded. Overall for Figures 3C and 3D , a total of 862 measurements were made. 350 (40.6%) were counted as ''wake,'' 186 (21.6%) were counted as ''sleep'' and 326 (37.8%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3E with stimulus, a total of 180 measurements were made. 80 (44.4%) were counted as ''wake,'' 38 (21.1%) were counted as ''sleep'' and 62 (34.4%) could not be assigned to ''wake'' or ''sleep'' and were excluded. For Figure 3E without stimulus, a total of 131 measurements were made. 32 (24.4%) were counted as ''wake,'' 71 (54.2%) were counted as ''sleep'' and 28 (21.4%) could not be assigned to ''wake'' or ''sleep'' and were excluded. To measure responsiveness to stimulation, the slope of the linear fit of the movement speed over the first minute of stimulation was used to measure the velocity increase. These velocity increase measurements were averaged for every worm and were subsequently averaged over all worms measured. Data was statistically compared using the Wilcoxon signed rank test for paired samples so as to compare sleep and wake responses. To test for homeostasis, RIS was optogenetically inhibited in arrested L1 larvae. flp-11::ArchT expressing larvae were starved for 24 hr in the microchambers and imaged for 3 hr. During the first hour, baseline data was recorded without illumination. For another hour the data was recorded under green light illumination. The third hour was recorded without green light. For statistical comparison (two-sample t test) between the condition with supplemented ATR and without ATR, we used averaged data ranging from 2.5 -7.5 min after the end of green light stimulation. To test for reversibility, arrested L1 larvae that express either sra-6::ReaChr or mec-4::ReaChr were used 24 hr after hatching in the absence of food. Each animal was imaged for 30 min and illuminated after 15 min for 1500 ms with green light. This was repeated up to 6 times with a delay of at least 1 hr to allow the worm to recover from the stimulus. The reversibility recordings were classified post hoc in ''awake'' or ''asleep.'' During each data point, the worm was classified as awake, when it spent less than 25% in a sleep bout during the 15min of baseline measurement and was classified as asleep when it spent at least 95% of the time in a sleep bout during the 3.5 min time interval before the optogenetic activation started. Data that could not get clearly classified with the above criteria were excluded from the analysis. Awake and asleep data was averaged for every worm to give one value for wake and one for sleep. Data was then averaged across all worms. For statistical comparisons (two-sample t test), we averaged data from the 5min after the end of optogenetic stimulation.
Image Analysis We extracted two parameters from the acquired images: 1) worm locomotion velocity: Two methods were used: A) The worm's head position was determined either manually or automatically with a home-made MATLAB routine that detects the position of RIS in the head of the animal, or the centroid of the entire body, similar to the one previously described and calculated with a conversion factor to obtain the velocity in mm/s. This method was applied for Figures 2, 3B , 4C-4F, 5G, S3E, and S6C. B) Sleep bouts were extracted using frame subtraction using a MATLAB routine similarly as previously described . This method was applied for Figure S4 . 2) RIS depolarization as measured by GCaMP3 intensity: Using the coordinates for RIS position as determined by the MATLAB routine, RIS GCaMP3 intensity was extracted from the images. The RIS GCaMP3 intensity was calculated as the mean of the 30 highest pixel intensities in an 11pixel x 11pixel square around the RIS cell body. Pan-neuronal nuclear GCaMP6s intensity was calculated as the mean of the 500 highest pixel intensities in a 69pixel x 69pixel region of interest around the center of the head neurons. The intensities were subsequently normalized to obtain DF/F. We analyzed sleep bouts in movies with 3 hr recording time. To score for sleep, the velocity data was smoothed using a 1 st degree polynomial local regression model over 25 time points using the in-build smooth function in MATLAB. To be scored as a sleep bout, it had to last at least 3min with a smoothed velocity below 0.5mm/s for adults and dauers and below 3mm/s for arrested L1 larvae. In L1 larvae in the presence of food, a sleep bout was defined as a smoothed locomotion velocity below 1.2 mm/s for at least 2 min. To be scored as sleep using frame subtraction, intensity counts had to be below 20% of the average intensity. These cutoff parameters were determined empirically . For sleep amount comparisons the time spent in a sleep bout was divided by the total time. For developing L1 larvae in the presence of food, lethargus was defined by the non-pumping period. As a representative L1 wake phase we took 3 hr before lethargus onset. For averaging RIS activity and velocity, data was aligned to the sleep bout onset. RIS activity and velocity starting 15min before bout onset until 30min after sleep bout onset were selected for display. To obtain a flat baseline, selections that have sleeping bouts within the 15min before the bout onset were excluded from the alignment. The averaged RIS activities 5min before and 5min after the bout onset were compared using the Wilcoxon signed rank test for paired samples. The displayed RIS DF/F data of individual animals was smoothed with a 5-point-running-average.
Lifespan and survival assays To isolate L1 larvae for lifespan and survival assays, eggs were synchronized using bleaching of a mixed population of worms and collected in standard M9 buffer . The worms were allowed to hatch and to arrest overnight. The animals were then kept in the dark, at 20 C on a slowly spinning rotator in 2 mL Eppendorf tubes. At defined time intervals, we took samples and transferred the worms to fresh seeded NGM plates. We scored after 5-20 min the percentage of living worms to measure survival. A worm was scored as alive, when it was able to move on the plate. To analyze recovery, we kept the worms on the plates for 2-4 days at 20 C and then scored the percentage of worms that had re-entered development and had grown to at least larval stage L3 after feeding. At least 50 worms were scored at each time point. For comparisons, the time points ''50% alive'' and ''50% recovering'' were defined. These values represent the days when the survival/recovering curves reach 50% surviving or recovering worms . For survival measurements of arrested L1s in the presence of food, FUdR was used to cause the arrest. FUdR was added to a final concentration of 50mM to the synchronized worm culture in M9 at 20 C at day 4. At day 5, at least 100 worms were pipetted to NGM plates seeded with E. coli and containing 50mM of FUdR, with a worm density of roughly 20 worms per plate. We kept the plates at 20 C in dark and scored every two or three days for viability. Worms were identified as dead if they didn't shown locomotion after several gentle mechanical stimulations by pick. Dead worms were removed from the plates. Living worms were gently transferred to fresh FUdR NGM plates if any fungi contamination appeared on the plates. Worms that had dried out after crawling off the agar surface were excluded from the analysis. For adult lifespan assays on food, a standard lifespan protocol was used . In brief, a synchronized worm culture was grown until young adulthood. At least 50 worms were transferred to freshly seeded NGM plates with a worm density of roughly 10 worms per plate. Worms were kept at 20 C in dark and scored every two or three days for viability. They were classified as alive if they showed locomotion or, if quiescent, responded to gentle mechanical stimulation exerted by a platinum wire pick. Living worms were transferred to fresh NGM plates every two days in the first two weeks to separate them from their offspring until they had stopped laying eggs. Worms that had dried out after crawling off the agar surface or that died after leaking out through the vulva were excluded from the analysis. The lifespan assays of starving adults were performed similar to the lifespan assay on food. The differences were that young adults were kept to feed on plates supplemented with 50mM FUdR for two days before they were transferred to food-free plates containing again 50mM FUdR. To correlate sleep amount with survival, we kept arrested larvae inside agarose microchambers without food at 20 C in dark. The microchambers with L1 larvae were prepared as described above. When the agar appeared dry after a few days, we re-moisturized the microchamber with 20mL of S-Basal containing 100u/mL Nystatin (Sigma Aldrich). Nystatin was added to prevent fungal growth. Worms were imaged after two days of arrest using DIC time-lapse microscopy with a frame rate of 0.1frame/s to quantify sleep. Using a home-made MATLAB script, the worm's centroid was determined and its velocity measured. The resulting plot was smoothed over 40 time points. To be scored sleeping, the worm's smoothened velocity had to be below 40% of the average velocity for at least 2min. The time spent sleeping was divided by the total imaging time, averagely for each worm from more than 35 hr of time lapse footage recorded between day 2 and day 4 after starvation. A linear fit was calculated from worms within the range: mean (lifespan) ± SD (lifespan) . The others were excluded as outliers.
Quantification of bacteria consumption To quantify bacterial consumption in FUdR-arrested larvae, FUdR was added to a final concentration of 50mM to the 3day synchronized worm culture for 24 hr. Worms were transferred in agarose microchambers filled with OP50 that had been transformed with a GFP plasmid (pFPV25.1) [34, 35, 80] . Food consumption was measured by quantifying GFP fluorescence over time. As a control we used bacteria-filled chambers that did not contain worms. With standard GFP filter sets, 20x objective, 490 nm light (with 2.00 mW/mm 2 intensity), 5ms exposure time and 50 EM gain we quantified GFP signals one and three days after microchamber Figure 1 . 1 Figure 1. Food Conditions Control Sleep Amount in C. elegans across Stages (A-I) Individual RIS calcium imaging and sleep measurements. RIS activity is shown in red and locomotion speed in black; blue shading shows sleep bouts as defined by a locomotion cessation threshold. At the onset of a sleep bout, RIS activated and locomotion ceased.(A) An adult worm feeding on OP50 bacteria with bacterial growth medium. (B) An adult worm feeding on OP50 without bacterial growth medium. (C) An adult worm feeding on dead OP50 with growth medium. (D) An adult that was starved for 24 hr. (E) An L1 larva in the presence of food, 8 hr after hatching and prior to lethargus. (F) An L1 larva before and during lethargus as defined as the non-feeding period (starting at 0 hr) prior to the molt in the presence of food. (G) An arrested L1 larva that was starved for 24 hr. (H) A 3-day-old dauer larva in the absence of food. (I) An adult feeding on OP50 without growth medium in the presence of dauer pheromone. (J-O) RIS activity increased significantly with locomotion cessation at the onset of sleep bouts in all conditions. The increase of D F/F of the calcium sensor signal was (J) 126.1% ± 20.2%, n = 24 worms, *p < 0.001 for adults on growing OP50; (K) 32.5% ± 5.4%, n = 20 worms, p < 0.01 for starving adults; and (L) 22.0% ± 0.9%, n = 14 worms, *p = 0.014 for developing L1 worms during lethargus. (M-O) 58.7% ± 4.9% (M), n = 33 worms, *p < 0.001 for arrested L1 larvae; 36.8% ± 7.9% (N), n = 13 worms, p = 0.002 for dauer larvae; 45.7% ± 19.8% (O), n = 12 worms, *p = 0.02 for adults on stationary OP50 with dauer pheromone. For all statistical comparisons, the paired Wilcoxon rank test was used. See also FiguresS1 and S2.
Figure 2 . 2 Figure 2. The Sleep-Active RIS Neuron Is Required for All Types of Physiological Sleep
Figure 3 . 3 Figure 3. Behavioral Quiescence during L1 Arrest Presents a Sleep State (A and B) To test for responsiveness to stimulation during quiescence in arrested L1 larvae (A), a blue light stimulus was given and locomotion velocity was measured. (B) Linear regression of locomotion speeds during the first minute of blue light irradiation was used to measure locomotion acceleration. Waking worms (red dotted line, red box) accelerated with 0.22 ± 0.02 mm/s 2 , whereas sleeping worms (blue dotted line, blue box) accelerated only with 0.11 ± 0.01 mm/s 2 (n = 42 worms; *p < 0.001; paired Wilcoxon rank test). (C-E) To test for sleep reversibility, nociceptive ASH neurons and mec-4 expressing mechano-sensory neurons were stimulated optogenetically with ReaChr and green light in sleeping arrested L1 larvae. In addition, arrested L1 larvae were stimulated with noxious blue light. RIS activity is shown in red (control without all trans-Retinal [ATR] in light red), speed in black (control without ATR in gray), and the fraction of sleeping animals in blue (control without ATR in light blue).
Figure 4 . 4 Figure 4. The Nutrient-Sensing Longevity Regulators IIS and AMP Kinase Regulate Starvation Sleep through RIS Activation
Figure 6 . 6 Figure 6. Sleep Counteracts the Progression of Aging Phenotypes during L1 Arrest(A and B) Body-wall muscle myosin deteriorates faster in aptf-1(À). (A) Shown is the head region of transgenic worms expressing GFP-tagged myosin (representative examples). After two days of starvation, muscle structure appears intact in both wild-type and aptf-1(gk794). After fifteen days of starvation, muscle myosin deterioration has progressed much more in aptf-1(gk794) compared to wild-type. Arrowheads point to gaps in muscle structure. (B) Quantification of muscle deterioration. 4 days: n > 10, N2: 1.1 ± 0.3; aptf-1(gk794): 2 ± 0.3, *p < 0.05; 12 days: n > 10, N2: 2.2 ± 0.5; aptf-1(gk794): 15.6 ± 1.9, *p < 0.001; 15 days: n > 10, N2: 7 ± 1.7; aptf-1(gk794): 39.1 ± 2.7, *p < 0.001, two-sample t test. (C and D) Mitochondrial fragmentation is increased in aptf-1(À). (C) Mitochondrial morphology appears normal in one day starved larvae, but the initially tubular mitochondria fragmented much faster in aptf-1(À) compared with the wild-type. Mitochondria in body wall muscles were labeled using GFP and were classified as tubular (M [tub.]), intermediately fragmented (M [int.]), or completely fragmented (M. [frag.]). Shown are the mid-body regions of worms, and arrows point to example mitochondrial structures or nucleus (N). (D) For the quantification of mitochondrial deterioration, the percentage of muscle cells displaying each level of fragmentation was determined (12 days-M (frag.): n > 20, N2: 8.5% ± 3.3%; aptf-1(gk794): 47.9% ± 5.9%, *p < 0.001; 14 days-M (frag.): n > 20, N2: 15% ± 3.4%; aptf-1(gk794): 56.1% ± 6.1%, *p < 0.001, two-sample t test). (E and F) Protein aggregation is increased in aptf-1(À). (E) Poly-Q35 was expressed in muscle, and the number of aggregates formed was scored. In one day starved L1, there were almost no detectable aggregates in both wild-type and aptf-1(gk794). Shown are the mid-body regions of worms with arrowheads pointing at aggregates. (F) The number of aggregates increased much faster in aptf-1(gk794) compared with wild-type (12 days: n > 20, N2: 1 ± 0.2; aptf-1(gk794): 3.4 ± 0.4, *p < 0.001; 14 days: n > 20, N2: 2.5 ± 0.4; aptf-1(gk794): 4.7 ± 0.7, p < 0.01, two-sample t test).
daf-16(mgDf50) I; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. This paper and Caenorhabditis Genetics Center HBR1709 HBR1753: wtfIs5[prab-3::NLS::GCaMP6s; prab-3::NLS::tagRFP] This paper, created from AML32 (Andrew Leifer lab) HBR1753 HBR1777: goeIs384 [pflp-11::egl-1::SL2-mkate2-flp-11-3 0 UTR, unc-119(+)]. This paper HBR1777 HBR1807: goeIs232[psra-6::ReaChr::mKate2-unc-54-3 0 UTR, unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. This paper HBR1807 HBR1808: goeIs251[pmec-4::ReaChr::mKate2-unc-54-3 0 UTR,unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. This paper HBR1808 HBR1830: daf-2(e1370) III; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)].
CF2570HBR2158: daf-16(mgDf50) I; aak-2(ok524) X; tdEx618[ppgp-1:: RFP+rol-6(su1006)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2:: mKate2-unc-54-3 0 UTR, unc-119(+)]. This paper and Masamitsu Fukuyama lab HBR2158 HBR2159: daf-16(mgDf50) I; aak-2(ok524) X; tdEx589[ pMF342, pmyo-3::GFP::aak-2::unc-54 3 0 UTR]+ppgp-1::RFP +rol-6(su1006)]. This paper and Masamitsu Fukuyama lab HBR2159 HBR2160: daf-16(mgDf50) I; aak-2(ok524) X; tdEx700[pMF380ppgp-1::GFP::aak-2::unc-54 3 0 UTR]+ppgp-1::RFP+rol-6(su1006)]. This paper and Masamitsu Fukuyama lab HBR2160 HBR2161: daf-16(mgDf50) I; aak-2(ok524) X; tdEx632[pMF308prgef-1::GFP::aak-2::unc-54 3 0 UTR+ppgp-1::RFP+rol-6(su1006)]. This paper and Masamitsu Fukuyama lab HBR2161 HBR2162: daf-16(mgDf50) I; aak-2(ok524) X; tdEx541[pMF312, pdpy-7::GFP::aak-2::unc-54 3 0 UTR+ppgp-1::RFP+rol-6(su1006)]. This paper and Masamitsu Fukuyama lab HBR2162 HBR2163: daf-16(mgDf50) I; aak-2(ok524) X; tdEx679[pKS19, paak-2::aak-2::GFP::unc-86 3 0 UTR+ppgp-1::RFP+rol-6(su1006)]. This paper and Masamitsu Fukuyama lab HBR2163 HBR2173: daf-16(mgDf50), aak-2 (ok524) X; goeEx720 [pflp-11::aak-2a::SL2-mKate::flp-11 3 0 UTR, unc-119(+) + myo-2::mCherry]; [ppgp-1::RFP+rol-6(su1006)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. This paper and Caenorhabditis Genetics Center HBR2173 HBR2151: daf-16(mgDf50), aak-2 (ok524) X; rrEx127[paak-2::aak-2+rol-6(su1006)].This paper and Richard Roy lab HBR2151 HBR2152: daf-16(mgDf50), aak-2 (ok524) X; rrEx191[psulp-5::AAK-2+rol-6(su1006)] . This paper and Richard Roy lab HBR2152 HBR2153: daf-16(mgDf50), aak-2 (ok524) X; rrEx123[pelt-2::aak-2+rol-6(su1006)] . This paper and Richard Roy lab HBR2153 HBR2154: daf-16(mgDf50), aak-2 (ok524) X; rrEx120[psur-5::aak-2+rol-6(su1006)] . This paper and Richard Roy lab HBR2154 (Continued on next page) e2 Current Biology 28, 3610-3624.e1-e8, November 19, 2018
N2: wild-type HBR227: aptf-1(gk794) II. HBR1361: goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. HBR1374: goeIs307[pflp-11::ArchT::SL2mKate2-unc-54-3 0 UTR,unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2unc-54-3 0 UTR, unc-119(+)]. HBR1709: daf-16(mgDf50) I; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 UTR, unc-119(+)]. HBR1753: wtfIs5[prab-3::NLS::GCaMP6s; prab-3::NLS::tagRFP] (created from AML32 (a gift from Andrew Leifer) by backcrossing 2x into N2) . HBR1777: goeIs384 [pflp-11::egl-1::SL2-mkate2-flp-11-3 0 UTR, unc-119(+)]. HBR1807: goeIs232[psra-6::ReaChr::mKate2-unc-54-3 0 UTR, unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2unc-54-3 0 UTR, unc-119(+)]. HBR1808: goeIs251[pmec-4::ReaChr::mKate2-unc-54-3 0 UTR,unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2unc-54-3 0 UTR, unc-119(+)].
K171: pmec- 4 : 4 :ReaChr::mKate2-unc-54-3 0 utr, unc-119(+) K172: psra-6::ReaChr::mKate2-unc-54-3 0 utr, unc-119(+) K214: pflp-11::ArchT::SL2-mKate2-unc-54-3 0 utr,unc-119(+) K216: pflp-11::SL1-GCaMP3.35-SL2::mKate2-unc-54-3 0 utr, unc-119(+) K281: pflp-11::egl-1::SL2-mKate2-flp-11-3 0 utr, unc-119(+) K358: pflp-11::aak-2a::SL2-mKate2-flp-11 3 0 utr, unc-119(+)
for tissue-specific rescue experiments. A B C D E F G H I Figure 5. Sleep Is Required for Surviving Larval Starvation-Induced Arrest Shown are representative experiments; additional replicates can be found in Figure S5. (A) Adult lifespans of feeding wild-type (blue) and aptf-1(gk794) (red) worms were not significantly different (mean lifespan, N2: 14.6 days, n = 44 worms; aptf-1(gk794): 15.3 days, n = 43 worms; p = 0.71; log rank test). (B) Lifespan of feeding wild-type (blue) and RIS(À) (yellow) adult worms shows no significant difference (mean lifespan, N2: 14.2 days, n = 94 worms; RIS(À): 14.1 days, n = 86 worms; p = 0.71; log rank test). (C and D) No significant difference of lifespan was detected in (C) starved adult aptf-1(gk794) compared to starved N2 (mean lifespan, N2: 29.8 days, n = 60 worms; aptf-1(gk794): 29.5 days, n = 63 worms; p = 0.65; log rank test) and (D) also not in starved RIS(À) (mean lifespan N2: 32.0 days, n = 85 worms; RIS(À): 30.6 days, n = 84 worms; p = 0.06; log rank test). (E) L1-arrested aptf-1(gk794) mutants and RIS ablated worms showed substantially reduced survival in the absence of food (time at which 50% of worms were still alive, N2: 20.7 days, n > 60 worms for each time point; aptf-1(gk794): 12.1 days, n > 60 worms; *p < 0.001; RIS(À): 9.4 days, n > 60 worms; *p < 0.001; significance shown from day 8; Fischer's exact test). (F) Arrested L1 aptf-1(gk794) and RIS-ablated worms showed a significant decline in the ability to re-enter development when fed (time at which 50% of worms were still able to recover, N2: 18.7 days, n > 60 worms; aptf-1(gk794): 11.4 days, n > 60 worms; *p < 0.001; RIS(À): 8.5 days, n > 60 worms; *p < 0.001; significance shown from day 8 for RIS(À) and day 10 for aptf-1 mutant; Fischer's exact test). (G) Sleep amount predicts starvation survival. Arrested starved L1 larvae were cultured in microchambers, and their sleep and survival were quantified. Data from individual worms within the range used for linear fitting (black line) are plotted as blue diamonds (data outside the range of the SD of the mean, including earlydying immobile individuals, were excluded; gray); R 2 = 0.21. (legend continued on next page)
TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains OP50 Escherichia coli B, Uracil auxotroph Caenorhabditis Genetics Center OP50 OP50 Escherichia coli that contains a GFP plasmid (pFPV25.1) Caenorhabditis Genetics Center OP50-GFP Chemicals, Peptides, and Recombinant Proteins FUDR, 5-Fluoro-2 0 -deoxyuridine Sigma-Aldrich F0503 Levamisole hydrochloride Sigma-Aldrich PHR1798 Experimental Models: Organisms/Strains N2: wild type Caenorhabditis Genetics Center N2 HBR227: aptf-1(gk794) II. HBR227 HBR1361: goeIs304[pflp-11::SL1-GCaMP3.35-SL2::mKate2- This paper HBR1361 unc-54-3 0 UTR, unc-119(+)]. HBR1374: goeIs307[pflp-11::ArchT::SL2mKate2-unc-54- 3 0 UTR,unc-119(+)]; goeIs304[pflp-11::SL1-GCaMP3.35- SL2::mKate2-unc-54-3 0 UTR, unc-119(+)].
Current Biology 28, 3610-3624, November 19, 2018
(legend continued on next page)
e4 Current Biology 28, 3610-3624.e1-e8, November 19, 2018
e6 Current Biology 28, 3610-3624.e1-e8, November 19, 2018
Current Biology 28, 3610-3624.e1-e8, November 19, 2018 e7
e8 Current Biology 28, 3610-3624.e1-e8, November 19, 2018
|
10.21203/rs.3.rs-1267005/v1
|
cc-by
| null | null |
openalex
|
Earth ssures are a type of major geological disaster that occurs worldwide. China has experienced some of the most serious earth ssure disasters in the world. The Yuncheng Basin is an important constituent basin of the Fenwei Basin in China where earth ssures are densely developed and caused severe damage. In particular, the impact of earth ssures on the seismic response of the site is still unknown and is an urgent problem that needs to be solved. Based on microtremor tests, three types of typical earth ssure sites in the Yuncheng Basin were selected for eld testing. Through spectrum analysis, the dynamic response characteristics of the earth ssure sites were determined. The results show that the dynamic response of the site is signi cantly affected by the earth ssures. The dynamic response strength of the site is the largest on both sides of the earth ssures, and it decreases and gradually stabilizes with increasing distance from the ssures. The impact range of the earth ssures on the hanging side is slightly longer than the heading side.Introduction Earth ssures are linear tensile ssures with a certain extension length that develop on the earth surface and may be accompanied by vertical dislocations. They are affected by various complex factors such as internal and external forces and human activities (Carpenter 1980 ). Earth ssures are widely developed around the world and occur in the United States, Mexico, Africa, Europe, and throughout most of China. Thus, earth ssure disasters are an important geological disaster that restricts the development of the national economy and seriously affects infrastructure construction (Qiao et al. 2015) . The existence and activity of earth ssures do not only cause direct damage to aboveground and underground structures, but when an earthquake occurs, the presence of earth ssures can change and even increase the seismic response of the site, causing the structures near the earth ssures to suffer severe damage or even be destroyed . Humans have been studying earth ssures for a century. The rst reported earth ssure was discovered in the Goose Greek oil eld in the Houston-Galveston area in the United States in the 1920s. At this time, some scholars believed that it was caused by oil exploitation (Snider 1927; Minor 1925 ). Since then, earth ssures have been reported in many areas of the southwestern United States and have caused considerable economic losses. A series of scholars have launched intense discussions of the formation mechanisms of earth ssures, which have resulted in several relatively systematic understandings. The main cause hypothesis states that the causes of earth ssures include tectonic genesis (Leonard 1929) , groundwater genesis (James, Langer and Kerr 1968; Fletcher et al. 1954) , and structural and groundwater compound genesis (Holzer 1980a, b) . Later, as amount of research, observations, and data on earth ssures increased, it was discovered that the formation mechanism of earth ssures is very complicated. Thus, the combined cause theory was generally developed. In addition to the United States, Mexico (Rojas, Arzate, and Arroyo 2002), Ethiopia (Laike 1998) , and other countries have also carried out research on earth ssures. Earth ssures are widely developed in China and cause severe damage. The earth ssures in the Fenwei Basin, the Hebei plain and Su-Xi-Chang area are most typical earth ssures (Fig. 1 ) (Wan et al. 2019). Among them, the earth ssures in the Xi'an area, which is located in the Fenwei Basin, are the earliest and most representative. Many scholars have studied the earth ssures in the Xi'an area and have proposed their own formation theories. The early theories mainly include the structural theory (Li et al. 1986 ), groundwater theory (Li 1994) , and combined cause theory (Yang and Wu 1986) . With further in-depth research, some scholars (Deng et al. 2007 (Deng et al. , 2013;; Peng et al., 2007; Peng et al. 2016 ) proposed a formation model for the Xi'an earth ssures, which involved the internal and external dynamic coupling of the extensional fault activity and water action in a regional extensional environment. Other areas in the Fenwei Basin, such as the cities of Datong, Linfen, and Yuncheng, are also severely affected by earth Furthermore, because there is no systematic measurement data on the seismic response of earth ssure sites, in order to study this problem, it is necessary to develop new ideas and appropriate methods. Microtremor is a kind of constant micro-movement that has no speci c seismic source and can be observed at any time, and its amplitude is generally only a few microns. The Microtremor studies originated in the 1950s. The Japanese scholars (Kanai and Tamaka 1954 ) quantitatively analyzed the nature of microtremor surface waves. After this, a series of scholars investigated the in-situ microtremor sources, formation mechanisms, and waveforms of microtremors (Aki 1957; Ohta et al. 1978; Kagami et al. 1986 Kagami et al. , 1982)) . In the late 1960s, Toksoz and Lacoss used a seismic network to separate, extract, and analyze the various periodic components of microtremor (Lacoss, Kelly, and Toksoz 1969; Toksoz and Richard 1968 ) and reported on the components of the different bands of the microtremor and their possible causes. In the 1990s, Nakamura proposed a new microtremor method (Nakamura 1989 ). This horizontal-to-vertical spectral ratio (H/V) method has gradually become the focus of the analysis of site dynamics around the world. Many scholars try to explain the theoretical basis of this method from different angles (Bard 1998; Konno and Ohmachi 1998) . In 1994, Lermo and Chavez-Garcia (1994) summarized the three main types of microtremor analysis methods and concluded that the H/V method can effectively eliminate the source effect. Since then, the H/V method has been widely used in the eld of engineering. Many scholars in China have also contributed to the microtremor theory and practical engineering applications (Guo and Song 2000; Guo, Xie and Yu 2002; Guo and Ren 2005) . A large number of studies have shown that microtremor movement is an e cient, economical, and convenient method for testing the dynamic characteristics of a site, which contain a large amount of site soil structure information that can be used as eld measurement data. Some scholars (Zhang et al. 2017; Chang et al. 2020 ) studied the dynamic effects of earth ssures on sites using microtremor measurement data and found that the existence of earth ssures does indeed aggravate the vibration intensity of the site. The Yuncheng Basin is an important part of the Fenwei Basin and contains with the most well-developed earth ssures in China. The earth ssures in the Yuncheng Basin have caused serious disasters. earth ssures have become the most important geological disaster in this area. Furthermore, the basin has a large population, developed agriculture, and industry, and the earth ssures not only cause direct damage to local roads and houses but also severely restrict the construction of urban infrastructure, and thus they restrict the economic and social development in the Yuncheng basin. Therefore, in this study, three types of typical earth ssure sites in the Yuncheng basin were selected, based on microtremor testing and analysis, the in uence of the earth ssures on the dynamic response of the site was determined, and the ampli cation effect and in uence range of the earth ssures on the dynamic response of the site were preliminarily determined. The results of this study provide theoretical support for the development of seismic forti cation and avoidance measures for the earth ssure sites in this area.
Regional Structure From north to south, the Fenwei graben system consists of the Datong Basin, the Xinding Basin, the Taiyuan Basin, the Linfen Basin, the Yuncheng Basin, the Weihe Basin, and several other large fault basins (Fig. 1 ). The Yuncheng Basin is located in the southwestern part of Shanxi Province and has a total area of 4885 km 2 . The Yuncheng basin is a Cenozoic faulted basin superimposed on the multi-cyclic superimposed Ordos Basin. The sedimentary strata have been deformed by multiple tectonic movements and have experienced multiple tectonic periods, such as the Yanshanian and Himalayan movements. A representative large rift basin formed later (Zhao et al. 2018). The basic framework of the Zhongtiao mountain uplift area and the Emei platform uplift area in the basin was laid by the strong tectonic movement in the Yanshanian period, and it was mainly affected by the Himalayan movement during the Paleogene. Large-scale fault block tectonic movement occurred in this area. The tectonic setting of the basin has changed from a compressive and twisting thrust fault to a tension and twisting normal fault, and the embryonic form of the basin has been modi ed since then. The main structural deformations preserved today include a fault system in the Himalayan strike-slip extensional background (Qiao et al.
2015). The main tectonic movement of the Yuncheng basin are faults. It was controlled by the Yanshan movement in the Mesozoic, the Himalayan movement in the Cenozoic, and especially the tectonic movement since the Late Cenozoic. The fault structure in the basin is developed. The main active fault in the basin is the large fault on the northwest side of Zhongtiao mountain, which is also the main controlling boundary fault in the Yuncheng basin (Chen 2016 ). There are eight active faults in the basin, with NE, NEE, and NNE strikes. They enclose the boundaries of the Yuncheng basin and the secondary structural units inside the basin, and they mutually restrict and control the structural framework of the basin (Zhao et al. 2018). The Yuncheng basin is bordered by the Emei Platform to the west, and the Zhongtiao mountains to the southeast. The NE-trending Mingtiaogang in the middle of the basin divides the Yuncheng basin into the Sushui river plain and the Qinglong river plain. The basin is an asymmetrical sag basin that is deep in the south and shallow in the north. The basin contains a large amount of Cenozoic strata, accounting for about 80% of the total area, and the thickness of the strata increases from the northeast to the southwest. The thickness of the Cenozoic strata in the basin is generally greater than 1000 m, and the Quaternary sediments are also more than 300m thick. This loose, thick sedimentary layer provided the material basis for the extensive development of earth ssures. In general, factors such as the uplift of the horst in the Yuncheng basin, the faulted basement, the overexploitation of groundwater, and the loose, thick sedimentary layer have laid the foundation for the development of earth ssures in the basin.
Characteristics Of The Earth Fissures In The Yuncheng Basin The development of earth ssures is controlled by tectonic movement. The earth ssures in the basin are mainly affected by the secondary structural units. The Yuncheng basin has been affected by the Himalayan tectonic movement since the Cenozoic. Due to the control of the faults at the northern foot of the Zhongtiao mountain, the basement has been in a faulted extensional environment. Under the strong tectonic activity of the Zhongtiao mountain fault, the secondary structural unit in the basin was controlled, which intensi ed the uplift of the block and caused the formation of the Mingtiaogang earth ssures. The dip slip extension of the hanging wall normal fault in the basin caused the tension shear cracking of the surface soil layer, which laid the structural foundation for the development of earth ssures in the basin. In addition, since the 1980s, the over-exploitation of groundwater has been severe in this area, causing the groundwater level to continuously fall and the falling funnel to expand year by year, which induced a large number of earth ssures in the Yuncheng basin. Thus, the Yuncheng basin has become the faulted basin with the largest number of earth ssures in Shanxi. The earth ssures in the basin are mainly distributed on top of and on both sides of the Mingtiaogang uplift within the basin, in the Zhongtiao mountain uplift to the southeast, and in the Emei platform front edge to the north (Fig. 2 ). The earth ssures in the Yuncheng basin are distributed along the boundaries of the geomorphology, are concentrated along the fault zone, and are associated with land subsidence. The earth ssures predominantly have NE strikes. According to the survey, the earth ssure disasters in the basin mainly occurred before 1980 and between 1995 and 2005. A total of 119 earth ssures (belts) have been developed in the study area. They mainly strike NE-NEE and are generally 100-2000 m long. The longest ssures are up to 5000 m. They are generally 0.05-0.5 m wide, and the widest can reach up to 2 m wide. The small earth ssures account for 45% of the earth ssures (length less than 500 meters); the medium earth ssures account for 28% (length between 500-1000 meters); and the large earth ssures account for 17% (length between 1000-5000 meters). There is only one giant earth ssure in the basin. It is located in the salt lake district and is 10 km long and 0.3-1 m wide.
2(a), (b)) . Earth ssure F 1 strikes NE, which is consistent with the active fault in the underlying bedrock. In plane view, earth ssure F 1 is longer, its overall shape is linear. In cross-section, a smaller number of secondary ssures on both sides of the main ssure can be seen, the vertical dislocation of the two discs near the surface of the main ssure is small, and the deep dislocation is large, showing the characteristic of a synsedimentary fault (Fig. 3 passes through Xinzhuang-Yuwang-Lizhuang and other places, and it is more destructive along strike. As can be seen from the cross-section shown in Figure 3 , the formation is composed of interbedded loess and silty clay, and the vertical dislocation increases with increasing depth (Fig. 3(c) ). Earth ssure F 5 is 5 km from the Zhongtiao mountain fault zone, making it closest of the ve earth ssures to the fault zone. Earth ssure F 5 starts in Yuguo and ends in Zhongwei. Its overall trend is NE55, which is consistent with the trend of the Zhongtiao mountains. It extends for about 2.3 km, and the vertical dislocation of the ground surface is 8-20 cm. Earth ssure F 5 appeared in 2000, and it entered a period of rapid development after 2007. It is active and destructive along strike (Fig. 2(c )), and it is still developing. The earth ssure is a tilt-slip tension crack with obvious horizontal extensional movement and vertical differential movement. The differential settlement on both sides of the earth ssure is obvious, and the maximum vertical dislocation is 25 cm. As can be seen from the cross-section shown in Fig. 3 , the formation is composed of sandy soil and silty clay. The stratum is relatively weak, and there are associated secondary ssures around the main ssure, which mainly appear in the heading side (Fig. 3(b) ). 4 Microtremor Tests And Analysis
Survey Line and Data Point Layout Survey lines were laid along the previously described earth ssures (F 1 , F 2 , and F 5 ). Two survey lines (S1 and S2) were laid in Banpo and Wucao for earth ssure F 1 , only one survey line (S3) was laid in Yuwang for earth ssure F 2 , and one survey line each was laid in Yuguo and Zhongwei (lines S4 and S5, respectively) for earth ssure F 5 . The sketch map of survey line layout as shown in Fig. 5 . The survey lines were laid perpendicular to the earth ssures. Each survey line contained 18 data acquisition points, and 9 measurement points were set on either side of the earth ssure, where A represents the hanging side, and B represents the heading side. The survey lines were about 60 m long (Fig. 4 ).
Equipment and Methodology The testing instrument for used for the microtremor was a high-sensitivity servo-type velocity network seismograph (CV-374AV) manufactured by Tokyo Sokushin Co., Ltd. (Fig. 5 ). The sampling frequency of the instrument is 0.1-100 Hz. It can monitor in three orthogonal directions of microtremor data at the same time. The testing instrument meets the requirements of the microtremor test. The tests were carried out at night when it was quiet and in good weather in order to avoid obvious vibration sources. The microtremor in the X, Y, and Z directions were measured at each measurement point, and each measurement point was monitored for more than 10 minutes. If pedestrians or vehicles passed by during the test, these details were recorded, and the affected time period was avoided as much as possible when selecting the data. According to the above requirements, the microtremor signal after the observation was a stable random process. The collected speed-time history curve from the reliable signal was intercepted and converted it into the acceleration time-history curve. The intercepted data were imported into the SeismoSignal program. After the preprocessing, including ltering and baseline corrections, was completed, Fourier spectrum, response spectrum, and Arias intensity analyses were performed.
Microtremor Analysis Of The Earth Fissure Sites Figures 6-10 show the results of the microtremor spectrum analysis of the ve survey lines. As can be seen from the gures, The Fourier spectral results show that the Fourier spectrum patterns of the same earth ssure sites are basically the same, while there are differences in the spectra from different ssure sites. The Fourier spectrum patterns of the F 1 earth ssure site are all single peaks spectra, with prominent main peaks, small spectral areas, and concentrated predominant frequencies (Figs. 6 and 7 ). The Fourier spectrum patterns of the F 2 and F 3 ssures are both multi-modal, with many secondary peaks and large spectral areas (Fig. 8 9 10 ). The Fourier spectra are consistent with the characteristics of the earth ssure sites, the soil layer of the F 1 earth ssure site is relatively hard, and the secondary ssures are less developed, while the F 2 and F 5 earth ssure cross-sections reveal that their soil layers are weak, and the secondary ssures are densely developed on both sides of earth ssure F 5 . Further analysis of the predominant frequencies derived from the Fourier spectra shows that the earth ssures have less effect on the predominant frequencies of the sites, meaning that the predominant frequencies of the same survey line do not change signi cantly with increasing distance from the earth ssures, and the ranges of the predominant frequencies on the hanging and heading sides are basically the same. From the results of the spectral analysis, it can be concluded that the predominant frequency of the F 1 earth ssure site is 6-7 Hz, that of F 2 is 3-5 Hz, and that of F 5 is 3-5 Hz. However, because of the different soil structures of different earth ssure sites, the predominant frequencies of the different sites are different. The predominant frequency of the F 1 earth ssure site is the largest, and the predominant frequencies of the F 2 and F 5 earth ssure sites are similar. The predominant frequency of the site is mainly determined by the soil structure of the site and the physical and mechanical properties of the soil, and the earth ssures have little effect on the frequency. The response spectra of the same earth ssure sites are basically the same. The response spectrum of earth ssure F 1 has a main single peak, with few or almost no secondary peaks. The main peak is prominent, and the predominant period is concentrated. The response spectrum of earth ssure F 2 also has secondary peaks, its spectrum area is larger, the predominant period is more concentrated, and the response spectrum characteristics are consistent with the characteristics of the sites.The response spectrum of earth ssure F 5 exhibits multi-peak development, with many secondary peaks and a large spectrum area. The Arias intensity is the curve of the energy accumulation at the measurement point over time. The difference between the energy of the different measurement points can be seen better from the nal accumulated energy. Based on the Arias intensity of the three earth ssure sites, the closer the measurement point to the earth ssure, the greater the energy accumulated at the measurement point. The accumulated energies of each site reached the extreme values at measurement points A1 and B1; and as the distance increased, the energy gradually decreased and nally stabilized. 6 Analysis Of Dynamic Effects Of The Earth Fissures
Ampli cation Effect The Fourier spectrum, the amplitude of the response spectrum, and the relationship between the Arias intensity and the distance from the earth ssure of each survey line are shown in Fig. 11 . Although the response intensities of the different sites are different, the measurement points near the earth ssures all exhibit a signi cant ampli cation effect, which gradually attenuates and stabilizes with increasing distance from the earth ssure. In order to more intuitively reveal the degree and scope of the impact of the earth ssures on the dynamic response of the site, the concept of the ampli cation factor is introduced here. Figure 11 shows that the amplitudes of the three measurement points on both sides of the earth ssure on each survey line are basically the same and tend to be stable. Therefore, the stable amplitude is de ned as the average of the amplitudes of these three measurement points, and the ampli cation factor is de ned as the average amplitude of each measurement point divided by the stationary amplitude. Figure 11 shows the attenuation curve and tting curve of the ampli cation factor with distance. As can be seen from the gure, the ampli cation effect of the earth ssure is signi cant in the area near the earth ssure. The ampli cation factor decreases and gradually becomes stable with increasing distance from the earth ssure. The ampli cation factor tends to 1, which also indicates that the in uence of the earth ssure on the dynamic response of the site has a limited range. Table 1 shows the extreme values of the ampli cation factors derived from the Fourier spectrum, response spectrum, and Arias intensity. As can be seen from the table, the extreme values of the hanging side ampli cation factor are 1.8-2.9, and the extreme values of the heading side ampli cation factor are 1.7-2.9. Using the same analysis method, the extreme value of the hanging side is slightly greater than or equal to that of the heading side, and the ampli cation effect of the hanging side is slightly greater.
Range of In uence As can be seen from the previously described attenuation pattern of the ampli cation effect, the earth ssure has a certain range of in uence on the ampli cation effect of the site. When the ampli cation factor attenuates to 1, it can be said that the site is no longer affected by the ampli cation effect of the earth ssure, and thus, a dynamic response zone can be delineated. Based on Figure 12 , the in uence range of the earth ssure ampli cation effect was obtained (Table 2 ). As can be seen from Table 2 , the impact range is about 20 m, and the ranges provided by the different methods are slightly different. Furthermore, for the same analysis method, the scope of the in uence of the hanging side is slightly larger than that of the heading side. From the perspective of seismic forti cation in actual engineering, buildings should not be constructed in areas affected by earth ssures. If they must be constructed in these areas, the seismic forti cation intensity of the building should be appropriately increased according to the corresponding ampli cation factor. Earth ssures are distributed around the world, causing serious disasters, affecting the safety and stability of the surrounding buildings on the earth ssure sites. However, until today, there are very few studies about the dynamic response of earth ssures. The current seismic forti cation standards do not take into account the impact of the dynamic response of earth ssures. Therefore, this study based on the typical earth ssure sites in the Yuncheng Basin, and demonstrated the effect of ssures on the dynamic response of a site through spectral analysis of microtremors. (1) The predominant frequencies of three typical earth ssure sites in the Yuncheng Basin are not signi cantly different with the distance of the earth ssures. The frequency is only determined by the site. The earth ssures have no signi cant in uence on the predominant frequency. (2) The earth ssures have a signi cant ampli cation effect on the dynamic responses of the sites. For all of the different sites, the strongest dynamic response occurred at the earth ssures, and the ampli cation factor is the most signi cant near the earth ssures. Both the response and the ampli cation factor decrease and nally stabilizes with increasing distance from the earth ssures. The extreme values of the ampli cation factor in the Yuncheng Basin are 1.8-2.9 on the hanging side and 1.7-2.9 on the heading side. The dynamic response of the hanging side is slightly larger or almost equal to that of the heading side. (3) The dynamic response of the site is only affected by the earth ssures within a certain range. The impact ranges of the hanging and heading sides in the Yuncheng Basin are slightly different, but the overall impact range is about 20 m. (4) In the actual seismic forti cation of an engineering project, building in the area affected by the earth ssures should be avoided as much as possible. If it cannot be avoided, the seismic forti cation intensity of the buildings in the area affected by the earth ssures should be increased according to the corresponding ampli cation factor. Fourier spectrum, response spectrum, and Arias intensity analysis results for line S3 Figure 9 Fourier spectrum, response spectrum, and Arias intensity analysis results for line S4 ssure disasters. Many scholars have studied the distribution characteristics and formation mechanisms of earth ssures in the Yuncheng Basin (Wu, Jiang, and Li 2003; Peng et al. 2007; Song et al. 2007; Meng et al. 2010; Xu et al. 2010; Chen 2016; Qiao, Peng, and Lu 2017; Zhao et al. 2018; He et al. 2019). Until now, research on the development, distribution characteristics, and formation mechanisms of earth ssures all over the world has produced very rich results, but our understanding of the dynamic effects of earth ssure sites is insu cient. Several studies have investigated the dynamic effects of earth ssures under earthquake loads based on numerical simulations (Liu et al. 2012; Fan et al. 2014), and others have used indoor simulation experiments to explore the in uence of earth ssures on the dynamic response of a site (Liu et al. 2014; Huang et al. 2018; Xiong et al. 2018; Mu et al. 2019; Xiong, Zhang, and Chen 2019), but the existing results are few and unsystematic and are not su cient to guide engineering practice. Therefore, in terms of seismic forti cation, the Speci cation for site investigation and engineering design on Xi'an ground fractures (DBJ61-6-2006) only regards the seismic effects of earth ssure sites as a general site threat in accordance with the Code for seismic design of buildings (GB50011-2010).
Figure 3 3 Figure 3 shows six typical earth ssures in the Yuncheng basin and the damage they have caused to the ground and buildings. Earth ssure F 1 (Pleistocene earth ssure on the southern margin of the Mingtiaogang uplift) is located in the salt lake district of Yuncheng. It is the only giant earth ssure in the Yuncheng basin. Earth ssure F 1 appeared in 1975. It has a long extension, large scale, and strong continuity, and is not controlled by the pavement, buildings, and roads along the route. It is 10 km long and crosses Taocun-Banpo-Wucao-Xincao and other villages, and it is very destructive along strike (Fig.
Figures Figure 1 1 Figures
Figure Figure 8
Figure 8
Table 1 The 1 extreme value of the ampli cation factor of each analysis method Hanging Side Heading Side Fourier 2.1 1.8 Response 1.8 1.7 Arias 2.9 2.9
Table 2 2 The in uence range of each analysis method Hanging Side Heading Side Fourier 21.2 20.0 Response 20.0 19.5 Arias 21.5 17.0 7 Discussion And Conclusions
|
10.21203/rs.3.rs-2200872/v1
|
cc-by
| null | null |
openalex
|
Background Pregnancy and early infancy are increased risk periods for severe adverse effects of respiratory infections. Aboriginal and/or Torres Strait Islander (respectfully referred to as First Nations) women and children in Australia bear a disproportionately higher burden of respiratory diseases compared to non-Indigenous women and infants. In uenza vaccines and whooping cough (pertussis) vaccines are recommended and free in every Australian pregnancy to combat these infections. We aimed to assess the equity of in uenza and/or pertussis vaccination in pregnancy for three priority groups in Australia: First Nations women; women from culturally and linguistically diverse (CALD) backgrounds; and women living in remote areas or socio-economic disadvantage.
Methods We conducted individual record linkage of Perinatal Data Collections with immunisation registers/databases between 2012-2017. Analysis included generalised linear mixed model, log-binomial regression with a random intercept for the unique maternal identi er to account for clustering, presented as prevalence ratios (PR) and 95% compatibility intervals (95%CI).
Results There were 445,590 individual women in the nal cohort. Compared with other Australian women (n = 322,848), First Nations women (n = 29,181) were less likely to have received both recommended antenatal vaccines (PR 0.69, 95% CI 0.67-0.71) whereas women from CALD backgrounds (n = 93,561) were more likely to have (PR 1.16, 95% CI 1.10-1.13). Women living in remote areas were less likely to have received both vaccines (PR 0.75, 95% CI 0.72-0.78), and women living in the highest areas of advantage were more likely to have received both vaccines (PR 1.44,).
Conclusions Compared to other groups, First Nations Australian families, those living in remote areas and/or families from lower socio-economic backgrounds did not receive recommended vaccinations during pregnancy that are the benchmark of equitable healthcare. Addressing these barriers must remain a core priority for Australian health care systems and vaccine providers. An extension of this cohort is necessary to reassess these study ndings.Introduction Pregnancy and early infancy are increased risk periods for severe adverse effects of respiratory infections. (1, 2) A decrease in immunity from physiological effects of pregnancy, (3) and age-associated immune immaturity in infants, exacerbated in infants born preterm, are among the reasons for these heightened infection risks. (2) Other risk factors include co-morbidities, living in remote regions, lower socio-economic status, inadequate housing, decreasing rates of exclusive breastfeeding, and limited access to culturally safe and appropriate, affordable health care. (4) To combat respiratory infections among these groups, inactivated in uenza vaccines (IIV) and pertussis-antigen containing vaccines (dTpa) are recommended during pregnancy and provided free to all Australian women, (5) however data to inform the equity of antenatal vaccination coverage are scarce. Aboriginal and/or Torres Strait Islander (herein respectfully referred to as "First Nations") women and children in Australia bear a disproportionately high burden of respiratory diseases compared to non-Indigenous women and infants. (6) As such, First Nations women are a priority for antenatal vaccination. Several studies incorporating First Nations women have suggested the coverage of IIV in pregnancy is low (range < 3-49%), however small samples sizes and biases (7) (8) (9) (10) precluded reliable estimates of coverage. We previously reported lower and declining proportions of IIV and dTpa vaccination coverage among First Nations pregnant women compared to non-Indigenous pregnant women. (11) Also, although over 30% of Australian women are born overseas, (12) data describing antenatal IIV and dTpa vaccination coverage in women from culturally and linguistically diverse populations (CALD) are scarce. One Australian survey conducted over a three-month period examined IIV and dTpa vaccination coverage in pregnant women from CALD backgrounds in Victoria (13) . Although these results are important, the small sample size (n = 370) and restricted location preclude generalisable estimates of vaccine coverage in CALD women from other Australian jurisdictions and do not allow evaluation of temporal trends. A population-based study using data from Western Australia (WA) and New South Wales examined childhood vaccination coverage and showed pockets of lower vaccine coverage in children born from CALD mothers. (14) It is not known whether these pockets of low vaccine coverage extends to antenatal vaccination. To our knowledge, no other Australian study has examined the in uence of living in a remote area, or the level of socio-economic variation upon IIV and dTpa vaccine coverage in First Nations, non-Indigenous and non-CALD (hereafter referred to as 'other Australian'), and CALD pregnant women. The primary aim of this study was to provide robust estimates of antenatal IIV and/or dTpa vaccine coverage across three Australian jurisdictions over six years, among 1) women who identi ed as First Nations, who were from CALD backgrounds, or other Australians, and 2) among women from different levels of socio-economic advantage/disadvantage and remoteness of living. These data together are essential to indicate the equity of the Australian National Immunisation Program (NIP) and guide future antenatal vaccination policy.
Methods
Study design and population Links2HealthierBubs (Links2HB) is a retrospective observational cohort study of mother-infants pairs. (15) The eligible cohort comprised mothers with a pregnancy >= 20 weeks gestation, between 01 Jan 2012-31 and Dec 2017 inclusive, in the Northern Territory (NT), Queensland (Qld), and WA. Collectively these jurisdictions encompass 5.92 million km 2 of land, incorporating the largest geographic land mass and diverse climatic variations in Australia, (16) with ~ 95,000 births annually, (~ 32% of Australia's total number of births). ( 17 )
Inclusions/Exclusions The unit of measurement for this study was individual women not individual pregnancies, therefore repeat pregnancies throughout the study period were not included. Women were excluded from the study if: they recorded a birth before 20 weeks gestation; had missing data related to gestation at birth; or birthed outside the study period.
Data collection methods and sources The cohort was created through the linkage of Perinatal Data Collections (PDCs) and immunisation registers and databases. These data sources are described in Supplementary box 1. A combination of deterministic and probabilistic record linkage was used to identify individual women and their pregnancies. Analysis of antenatal IIV coverage involved the whole study period (2012-2017) whereas dTpa was analysed from 2015 onwards following implementation of funded vaccination programs. (18) The key variables used to estimate vaccination coverage were; infant date of birth, gestation in weeks at the time of infant birth, gestation in weeks at the time of vaccination, and trimester of pregnancy in which maternal vaccination occurred. Vaccination in pregnancy (antenatal vaccination) was de ned by documented receipt of an IIV and/or dTpa vaccine between the date of conception and the date of infant birth. Participants with no IIV and/or dTpa vaccination data during a pregnancy were considered 'unvaccinated in pregnancy'. First Nations status, ethnicity, socio-economic advantage and geographic variation First Nations status was determined from identi ers located in PDCs and hospital admissions data. In WA, the data linkage branch also identi es Indigeneity through a validated algorithm implemented by the WA Department of Health, for any individual with at least one record in a government administrative dataset. (19) Ethnicity and mother's country of birth variables recorded in the PDCs were used to categorise women from CALD backgrounds. Country of birth classi cations were from Standard Australian Classi cation of Countries (SACC), 2016 Australian Bureau of Statistics (20). A composite variable was created to classify women as 'First Nations' if they identi ed as 'Indigenous' AND were classi ed as 'Aboriginal and/or Torres Strait Islander' in the ethnicity variable; b) CALD if they did not identify as First Nations AND were not born in Australia AND were not classi ed as Caucasian in the ethnicity variable; and c) 'Other Australian' if they were Australian born and did not identify as First Nations AND were classi ed as 'Caucasian' in the ethnicity variable (Supplementary box 2). The Index of Relative Socio-economic Advantage and Disadvantage (IRSAD); derived from the Socio-Economic Indexes for Australia (SEIFA; Australian Bureau of Statistics 2016) index, (21) was used to de ne socio-economic advantage and disadvantage. The score includes measures of economic, educational and social domains and is ranked on a scale from 1-10, with one being the most disadvantaged and 9 and 10 being the most advantaged. The Accessibility Remoteness Index of Australia (ARIA) (22) was used to identify remote and non-remote living participants, and hence their accessibility to health care services. Remote and very remote categories were combined to enhance statistical power. The mother's recorded postcode of residence at the time of infant birth was used to establish their SEIFA and ARIA indexes.
Data analysis Pregnancy, antenatal vaccination, socio-economic and geographic characteristics were calculated for individual First Nations, Other Australian and CALD women and are expressed as proportions. A generalized linear mixed model (GLMM), log-binomial regression with a random intercept for the mother's ID to account for clustering of multiple pregnancies for the same mothers was used. To determine the relative in uence of a) living remotely (ARIA) and b) socio-economic disadvantage (IRSAD/SEIFA) on antenatal IIV and dTpa vaccine coverage, we presented the unadjusted prevalence risk (PR) and 95% compatibility intervals (95% CIs). (23) Variables statistically (5% signi cance level in univariable analysis) or speculatively associated with maternal vaccination coverage were included as covariates in a multivariable GLMM and reported as adjusted prevalence ratios (aPR) with 95% CIs. Not living remotely and SEIFA 1 were used as the reference groups. Data were analysed using Stata statistical software v.17.1 (StataCorp, College Station, Texas).
Ethics and governance Multi-jurisdictional ethics committee approvals were gained from WA Department of Health (HREC 2016/56), Curtin University (HRE2017-0808), Menzies School of Health Research (HREC 2018-3199), Queensland Health and Royal Brisbane and Women's Hospital (HREC/2018/QRBW/47660), and WA Aboriginal Health Ethics Committees (HREC 889). ( 15 )
Results There were 445,590 individual women in the nal Links2HB cohort (Figure 1 ), comprising 29,181 First Nations, 322,848 Other Australian and 93,561 women from CALD backgrounds. * Australian born women, who did not identify as First Nations and were classi ed as 'Caucasian' in the variable 'Ethnicity' Demographic characteristics of participants by First Nations status and ethnicity are shown in Table 1 . Table 1 : Demographic characteristics of LinksHealthierBubs participants by Indigeneity and ethnicity, Australia 2012-2017. ABBREVIATIONS:CALD, Culturally and linguistically diverse; SEIFA, Socio-Economic Indexes for Australia * Women who were Australian born, who did not identify as First Nations and were classi ed as 'Caucasian' in the variable 'Ethnicity' † Qld data only §Based on Accessibility Remoteness Index of Australia ¶ Ranking out of 10, where 1=highest level of disadvantage, and 10=most advantaged Overall, 69,670 (16%) received an IIV during pregnancy, 125,023 (43%) received dTpa and 53,861 (18%) received both vaccines during pregnancy. Respective coverage of IIV among First Nations, Other Australian and CALD women was 14%,15% and 19%; for dTpa this was 31%, 44% and 43%; and for both vaccines, 13%, 18% and 21% (Supplementary Table 1 ). Compared to other Australians, First Nations women were less likely to have received any of the recommended vaccines during pregnancy: dTpa; PR 0.70, 95%CI 0.69-0.71) and both vaccines; PR 0.69, 95%CI 0.67-0.71, and women from CALD backgrounds were more likely to have received IIV (PR 1.24, 95%CI 1.22-1.25) and both vaccines (PR 1.16, 95%CI 1.10-1.13) seen in Table 2 . Table 2 : Prevalence of antenatal vaccine coverage among Links2HealthierBubs cohort, 2012-2017. Factors that positively predicted antenatal vaccine coverage for the overall cohort were; increasing maternal age at birth, birthing in a private hospital, parity, attending antenatal care during the rst trimester, and increasing socio-economic advantage (SEIFA) (Table 2 ). Factors positively associated with vaccine coverage within the population groups were similar to those overall, apart from increasing maternal age among First Nations women (PR 0.82, 95%CI 0.71-0.93), and relative to the other jurisdictions, antenatal vaccine coverage in the NT was consistently lower (Supplementary Table 2 ).
Remoteness and socio-economic in uence Coverage of antenatal IIV was similar (13-19%) between population groups regardless of remoteness of living, however coverage of dTpa and both vaccines were 10-20% lower among First Nations women, particularly for those living remotely (Supplementary Figure 1 ). This trend was consistent across each jurisdiction (Supplementary Figure 2 ). Univariable and multivariable models con rmed statistically that overall, living remotely detracted from antenatal vaccination coverage: IIV PR 0.88 (95% CI 0.85-0.90); dTpa PR 0.75 (95% CI 0.73-0.76); and both vaccines PR 0.75 (95% CI 0.72-0.78) (Table 1 ). This trend remained consistent among all groups (Supplementary Table 2 ). Antenatal vaccine coverage increased with socio-economic advantage. The highest proportions of vaccine coverage were in the SEIFA 10 decile compared to women from areas of highest disadvantage (SEIFA deciles 1 and 2) shown in Figure 2 . This positive association was most notable for coverage of dTpa, and both IIV and dTpa vaccines among First Nations women in SEIFA 9 decile: IIV (PR 1.28, 95%CI 1.05-1.57); dTpa, (PR 1.33, 95%CI 1.20-1.47); and both IIV and dTpa; (PR 1.36, 95%CI 1.14-1.62) . The trends were consistent across each group (Supplementary Table 2 ), and each jurisdiction (supplementary gures 3a-3c).
Discussion We identi ed overarching inequity in coverage of antenatal vaccinations among First Nations women, and in women who lived in remote areas, and women who lived in areas of higher socio-economic disadvantage. Other Australian women and CALD women living in major cities were more likely to have received the recommended and funded vaccines in pregnancy, and women from lower socio-economic backgrounds were less likely to have received vaccines. We also con rmed that younger women who birthed in public hospitals and who attended antenatal care later during pregnancy were less likely to have received antenatal vaccines. These geographic, social and nancial access issues demonstrate shortcomings in our current health systems and NIP, and highlight the need for more dedicated resources and training to address the inequity. As with other studies, we saw vaccination coverage improve over the years of the study, (24) particularly following the implementation of the antenatal dTpa program, but despite surveys indicating a high willingness of First Nations women to be vaccinated in pregnancy if offered, (7, 25) coverage of antenatal IIV and dTpa vaccination remained low. The gap between willingness and coverage is a potential physical and nancial health systems access issue. Designing antenatal services speci cally for younger mothers and promoting young parent programmes similar to some existing models may be one way to increase antenatal vaccination coverage among both younger and First Nations mothers. (26) Midwifery group practices co-designed with First Nations peoples for First Nations families, and antenatal care provided by First Nations health workers and midwives have demonstrated improved antenatal care attendance and equitable or better outcomes among First Nations mothers and infants compared to other Australians in mainstream care. (27, 28) However, this has not translated into practice for antenatal vaccination coverage in all jurisdictions as yet. Data from a 2021 clinical audit within a major Qld public hospital showed both antenatal IIV and dTpa coverage remains considerably low among First Nations mothers (~17% for IIV and 26% for dTpa) compared to non-Indigenous mothers (39% for IIV and ~61% for dTpa). (29) Where First Nations or CALD focused midwifery models of care are not in place, more attention should be given to developing culturally safe clinical practice, and improving vaccine education and service delivery to avoid systemic barriers to equity such as racism and discrimination. (30) Strategies to increase antenatal vaccination coverage could include the use of appropriate language and interpreters in healthcare provider service settings.
Strengths and limitations Our data include the largest linked cohort of First Nations mother-infant pairs across three Australian jurisdictions, and includes urban, rural and remote-living representation. The WA validated algorithm used to identify maternal First Nations status across multiple datasets strengthens the accuracy of these data. Our data are also the rst to describe antenatal vaccination status by areas of accessibility to services and socio-economic advantage and disadvantage based on individual postcode level data. Our data did not include First Nations status or ethnicity of infants. We acknowledge that including Indigeneity only of the mother underrepresents First Nations infants of First Nations fathers, and that the mother may choose to not identify as First Nations due to risks of receiving poorer care. (30) Our large sample size of First Nations mothers however, affords good generalisability for the three jurisdictions included in our study. We also acknowledge potential limitations around using the variables 'country of birth' and 'ethnicity' as an imperfect way of assessing English language capacity and health literacy. We also acknowledge potential unknown errors of vaccine reporting.
Conclusions Our data demonstrated signi cant inequity in antenatal vaccination among First Nations Australian families, and families who live remotely and/or from lower socio-economic backgrounds. Given that the known increased risk factors for acquiring in uenza or pertussis infections are living in remote regions, lower socio-economic status, inadequate housing, and limited access to culturally safe and appropriate, affordable health care, vaccination against these infections remains a key public health strategy in preventing severe disease in pregnancy and early infancy. Systematically monitoring vaccine coverage, and strategies that ensure vaccines are offered and provided to women equitably alongside other quality healthcare during pregnancy are required. This is a core responsibility of Australian health care systems and vaccine providers.
List Of Abbreviations Figure 1 . 1 Figure 1. Participant ow diagram of unique Links2HealthierBubs study participants by ethnicity and jurisdiction, Australia, 2012-2017.
Figure 2 : 2 Figure 2: Antenatal vaccination by Indigenous status, ethnicity and socio-economic variation
Figures Figures
Figure 1 Participant 1 Figure 1
Table 2 2 All methods were carried out in accordance with relevant guidelines and regulations, and protocols were approved by ethics committees. Due to the study design and sample size of this study, a waiver of informed consent was sought and granted by the following ethics committees: WA Department of Health (HREC 2016/56), Curtin University (HRE2017-0808), Menzies School of Health Research (HREC 2018-3199), Queensland Health and Royal Brisbane and Women's Hospital (HREC/2018/QRBW/47660), and WA Aboriginal Health Ethics Committees (HREC 889). Overarching ethics approvals were gained from each of the abovenamed committees. Women who were Australian born, who did not identify as First Nations and were classi ed as 'Caucasian' in the variable 'Ethnicity' † Qld data only § Based on Accessibility Remoteness Index of Australia ¶ Ranking out of 10, where 1=highest level of disadvantage, and 10=most advantaged Prevalence of antenatal vaccine coverage among Links2HealthierBubs cohort, 2012-2017.Abbreviations: PR, prevalence ratio; IIV, inactivated in uenza vaccine; CALD, Culturally and linguistically diverse; SEIFA, Socio-Economic Indexes for Areas; dTpa, diphtheria-Tetanus-acellular pertussis vaccine. * Qld data only †Data restricted to >2014 in line with recommendations for dTpa in pregnancy IIV, inactivated in uenza vaccines, dTpa, diptheria-tetanus-acellular pertussis vaccine ABBREVIATIONS: CALD, Culturally and linguistically diverse; SEIFA, Socio-Economic Indexes for Australia *
|
10.3389/fpubh.2022.725256
|
cc-by
| null | null |
openalex
|
Background: On May 3, 2016, residents of Fort McMurray Wood Buffalo, Alberta were evacuated due to an uncontrolled wildfire. The short-notice evacuation had destabilizing consequences for residents, including changes in routines, loss of control, and increased uncertainty. These consequences were especially detrimental to women who were pregnant or pre-conception during the evacuation. Pregnant and pre-conception women are particularly susceptible to a vast range of negative consequences during and post natural disasters, including elevated stress and higher incidence of pregnancy complications including gestational diabetes mellitus, pregnancy induced hypertension and C-section. The aim of this study was to understand the experiences, perceived stress and resilience of women who were pregnant during the wildfire. As well as to explore potential interventions to promote the health and enhance resilience of pregnant women and to assist in recovery after exposure to a natural disaster or other traumatic events. Methods: A qualitative thematic analysis of 16 narratives penned by pregnant women and recounted in Ashley Tobin's compilations 93/88,000 and 159 More/ 88,000: Stories of Evacuation, Re-Entry and the In-Between was conducted. Results: Analysis revealed five key themes: (1) experience of stress responses due to personal and external factors, (2) social connectedness and support as a facilitator of resilience, (3) performance of resilience-enhancing activities, (4) the roles of pregnancy and motherhood in the experiences of loss and resilience, and (5) the importance of home.
Conclusion: Pregnant women have unique barriers that may negatively impact them during a natural disaster or other form of stressful event. They may benefit from assistance with navigating role transition during pregnancy, training in stress management strategies, and writing interventions to build resiliency and begin the process of recovery from trauma.INTRODUCTION An unrelenting, unpredictable wildfire engulfed Fort McMurray Wood Buffalo (FMWB), Alberta in 2016, prompting the shortnotice city-wide evacuation of approximately 88,000 people (1) . Beginning on May 1, 2016, the fire remained uncontrolled until July 5, 2016, and burned nearly 570,000 hectares of land. An estimated $6 billion in commercial and personal damages resulted due to the destruction of approximately 2,400 homes and businesses (2) . Residents in specific areas were able to return to their homes in June 2016, while others who lost their homes were unable to return until the spring of 2017. The economic impact on the community was not the only destabilizing consequence residents of FMWB experienced. Residents received little warning in advance of the wide-scale evacuation, which resulted in significant disruption of routines, loss of control, and increased uncertainty, potentially increasing stress and anxiety (3) . Despite the disruption, affected individuals must find a way to continue moving forward. In 2016, 55,595 babies were born in Alberta. While it is unknown how many of these women and infants were impacted by the FMWB wildfire specifically, pregnant women experiencing disasters are in a unique position, as unexpected traumas can impact their developing fetus (4) .
Impacts of Disaster Exposure to Individuals The outcomes of disaster exposure are immensely complex and may take months or years to conclude (5) . Individuals exposed to disasters may experience increased observable stressors and develop subjective beliefs regarding cause and effects of the event (5) . Research into impacts of natural disaster exposure consistently indicates increased disturbances in social and psychological well-being, frequently influenced by an individual's coping styles in response to stressful events (6) . Women and children exposed to natural disasters experience increased stressors and a vast range of negative consequences including mental health concerns such as post-traumatic stress disorder (PTSD) (7) (8) (9) . Specific to FMWB, research by Moosavi and colleagues (10) found increased rates of probable PTSD, depression and anxiety as compared to self-reported prevalence prior to the wildfire in Fort McMurray Primary Care patients. Brown et al. (11) indicated similar increases in mental health concerns, with nearly half of student study participants meeting criteria for one or more probable diagnoses including depression, anxiety, PTSD or alcohol/substance abuse. Natural disasters can disrupt an individual's occupational performance and ability to perform daily activities within their environment. Experiencing a natural disaster during pregnancy adds a layer of complexity. An environmental event may upset a pregnant woman's routines, roles, and occupations during a very significant and meaningful time. Additional barriers, such as limits to mobility in pregnancy, may enhance the challenges caused by an environmental event. As stress due to unexpected trauma during pregnancy can have long-term impacts that can compound across generations (4), reducing these negative consequences is of critical importance. When combined with the vulnerability of exposure to a natural disaster, the intersection between pregnancy and trauma creates a form of double jeopardization (12) . As such, pregnant women who experience a natural disaster represent a vulnerable population, highlighting the importance of understanding women's experiences of pregnancy during traumatic events. Knowledge about how the women experienced stress and pregnancy throughout wildfire and evacuation can inform interventions to support women who have experienced trauma during pregnancy, as well as pregnant women who may experience a future traumatic event.
Prenatal Experience While pregnancy and childbirth are incredibly significant, personal, and spiritual experiences (13) , they cause changes in one's body, mind, relationships and routines and are associated with risks to a woman's physical, mental, and emotional well-being. Physical risks and complications include anemia, gestational diabetes mellitus, and hypertension (14) . Mental health challenges can also arise. These health risks can impact the developing fetus, with the average rate of fetal deaths per 1,000 total births ranging from 7.5 to 8.1 in 2005-2014 (15) . Preterm births during this same period were reported to occur in approximately 8 percent of live births, and very early births (prior to week 32) were found to make up 1.2 per 100 live births. In addition to changes in physical appearance, women may also experience changes in their identity, roles and occupations (16) , especially during their first pregnancy (17) . Changes in relationships may also occur as a new understanding of self and the roles of others are developed (18) . Pregnancy may facilitate greater intimacy in significant relationships or become a source of tension. Roles, occupations, and environments are important during pregnancy and are significant to understanding women's experiences of a natural disaster and their subsequent loss and grief. Pregnancy represents a transitional period (19) , despite the continuation of daily routines and occupations (13) . Engaging in normal routines and occupations, however, may become challenging during pregnancy and the post-partum period (19) , with mobility and sleep becoming challenging. Experiencing a natural disaster during pregnancy can further alter an individual's occupational performance and their ability to perform daily activities. In addition to the changes in occupation that women experience during pregnancy, they also take on new occupations specific to pregnancy and birth, including preparing for delivery, transitioning to family expansion, and caring for the baby (19, 20) .
Prenatal Maternal Stress and Child Development Experiencing stress during pregnancy increases the risk of longterm effects to both mother and child (21) (22) (23) (24) . Research further indicates that parental trauma can be transmitted to offspring, with outcomes dependent on a complex interplay of biological, familial, and cultural systems (25) . Pregnancy outcomes of women who experienced Hurricane Katrina showed a correlation between elevated stress and higher incidence of pregnancy complications including gestational diabetes mellitus, pregnancy induced hypertension and C-section (26) . Stress responses such as loss of sleep or appetite, which may affect maternal health, can also be attributed to environmental events (27) . Objective prenatal maternal stress (PNMS) can affect the development of the baby in utero and have negative impacts on birth weight (24, 27) . In an Australian study on birth outcomes following a bush fire, O'Donnell and Behie (22) found an increase in preterm births and babies born with low birth weight in comparison to previous years. PNMS has also been shown to impact childhood development. Research on the impacts of PNMS experienced by pregnant women during a flood was found to be negatively correlated with infant problem solving and personal-social skills at 6 months of age (23) . Project Ice Storm (28) , a research initiative following the Quebec ice storms of 1998, examined the long-term implications of prenatal exposure to a natural disaster on childhood development. PNMS was found to be related to decreased bilateral coordination and visual motor integration in children at five and a half years of age (21) . The impacts of maternal stress may be influenced by how the stress is perceived. Several studies have explored this relationship through examining the impacts of objective or subjective stress on both parent and child. Research by Cao et al. (21) found high maternal subjective distress was associated with greater negative impacts on motor function in children. Conversely, low subjective distress showed significant harmful effects when stress was high (21) . Simcock et al. (23) found that objective maternal stress measures were more predictive of decreased problem-solving skills in infants at 6 months. Based on research with rats, Yao et al. (4) suggested that experiencing a stressor, including exposure to natural disaster during pregnancy, may be predictive of chronic disease in later life. This study also found that prenatal stress compounded across generations and was a determinant in future maternal health (4) . As stress due to unexpected trauma during pregnancy can have long-term impacts that compound across generations (4), reducing these negative consequences is of critical importance. Exposure to a natural disaster in-utero may affect infant and childhood development, create long-lasting impacts on wellbeing, and impact future maternal health of infant girls. Understanding the experience of coping with stress during pregnancy throughout the wildfire and evacuation can inform interventions to support women who have experienced trauma during pregnancy, as well as pregnant women who may experience a future traumatic event. These findings substantiate the importance of further research in this field.
Potential Intervention Given the potential long-term physical and mental health outcomes trauma survivors and their offspring may encounter (25) , it is imperative that potential interventions be explored. Chen et al. (6) found that elements of emotion-focused coping, including positive reframing, acceptance, and emotional support were protective factors, reducing the subjective distress for pregnant women in flood-related natural disasters. Writing interventions provide an adaptive method for navigating life transitions and coping with traumatic and stressful events and can portray features of emotion-focused coping throughout narratives (29, 30) . A meta-analysis on writing therapy found writing to be an evidence-based and efficient intervention for individuals experiencing post-traumatic stress (31) . Horsch et al. (32) found that an expressive writing intervention was linked with decreased symptoms of depression and PTSD in mothers of preterm infants. Writing is efficient as it is cost-effective and does not require as much therapy time as other interventions (31, 32) . Studies have demonstrated that the task of writing is an effective intervention during times of stress. In a metaanalysis on writing therapies, van Emmerik et al. (31) indicated writing was an efficient and evidence-based intervention for populations experiencing post-traumatic stress, with decreased symptoms of post-traumatic stress and depression resulting. At times expressive writing is an intervention prescribed for persons experiencing times of stress and at other times, they spontaneously take up writing as a way of recording their thoughts and feelings in the moment.
OBJECTIVE This study sought to understand the experiences of women who were pregnant during or shortly following the Fort McMurray wildfire, particularly the perceived stress and resilience they experienced during the wildfire, evacuation and re-entry. This knowledge may help inform future interventions to enhance resilience, promote health, support post-traumatic growth and reduce the harmful effects of natural disasters and other traumatic stressors.
METHODS A qualitative secondary analysis of expressive writing using thematic analysis was conducted examining narratives written by 16 women who were pregnant during the 2016 FMWB wildfire. The narratives were captured in two books compiled by Ashley Tobin -93/88,000 (33) and 159 More/ 88,000: Stories of Evacuation, Re-Entry and the In-Between (34) . Convenience sampling was utilized by the author to obtain the written entries. Thematic analysis (deductive and inductive) was conducted using methods adapted by Braun and Clarke (35) . Graduate-level researchers transcribed the excerpts into NVivo 12. Data was coded and themes identified. All data was coded twice to increase consistency in the process and discrepancies were resolved through discussion. Emerging themes and patterns were analyzed, consolidated and refined and supporting quotes isolated.
RESULTS Based on the analysis of the women's narrated experiences of pregnancy during the evacuation, five key themes emerged: (1) experience of stress responses due to personal and external factors, (2) social connectedness and support as a facilitator of resilience, (3) performance of resilience-enhancing activities, (4) the roles of pregnancy and motherhood in the experiences of loss and resilience, and (5) the importance of home. Further Experiencing significant emotional responses. "The fire is all around us. It's like we were watching a movie or were stuck in a nightmare. It's like our whole world is falling down on us. We can feel the heat from the fire at our house and ash is falling from the sky. We're all in shock and can't move." "They told me to head to [company], and I would be able to at least get an EMT... to assist me. I just started to cry in total disbelief of what was happening. It seemed unreal." "Being under mandatory evacuation meant to me that I had to pack my stuff and get the hell out of there. I closed the windows, turned off the fans and AC, and grabbed the weirdest mix of stuff ever in my bag. I dumped the contents of our safe into my suitcase, the oddest mix of clothes with no underwear or socks, my secret cash stash, some water, a knitting project, a flask of water, and my husband's birthday presents." "As we started to get closer to town, it hit me. This is real life. My sister-in-law called back and said, "Don't panic, stay calm. We can't have you go into labor." I was doing fine until that call. I completely lost it." Loss of sleep and focus. "We were all so tired but couldn't sleep. Thinking about all the things we left behind and would never see again." "I thought I was OK. I was worried about my husband, I had dreams where he was being burned alive along with my unborn baby. He never stopped working and I didn't get to speak to him properly until May 5th. I was just glued to my phone and the TV, like a zombie." Pregnancy/labor complications. "My OBGYN finally explained to me that extreme stress can cause a baby to stop growing. There are medical terms that I researched and googled like crazy. Intrauterine growth restriction, placental insufficiency, small for gestational age." 1.2 Common fears were experienced. Separation from family/significant others. "That moment I had to leave my daughter + husband behind so I could get to Albian Sands quicker, it made me feel sick." "I freaked out. I was pregnant and alone with my daughter, literally running for my life down Abasand hill. My husband was at work trying desperately to come to us. I felt there was a possibility I may never see him again until eternity." Losing their home and possessions. "While waiting in line, I break down bawling my eyes out and my fiancé says "Are you okay? What's wrong?". Through my tears I say, "That could be the last time we see our home. We have a 19-month-old little girl, I'm 31 weeks pregnant and right now we may be homeless." "As we are coming up Beacon Hill I couldn't even look out the window. I'm sitting there thinking "what if we don't get to go back? I forgot so much stuff!" This is our home we could be losing along with thousands of other people." "That's when everything sank in. Will I see my home again? I didn't grab any baby items! I just had my baby shower!!" Transportation. "We all make the decision to try and head south once we get the chance. We knew north would not have enough accommodations for everyone and that we definitely wouldn't have enough gas to get us there." "As I was figuring everything out, I realized I had only 88 km to the tank. What if I get stuck in traffic? How would I get out of here?" "We headed down the one way on the wrong side of the road toward the camps. The highway was gridlocked. I can remember my husband saying, "One quick shift of the wind + were all wiped out." Pure terror set in as we waited in traffic for 2 hours." The well-being of the baby. "We weren't sure what our plan was yet but it didn't take long to decide the new smoky conditions wouldn't be great for our 48-hour old baby." "The first night, I was in so much pain from my C-section and was still trying to come to terms with what we just went through. I spent the night knelt on the floor hunched over the bed, crying, trying to sleep, and worried that something was going to happen to my baby." descriptions of themes with supporting quotes are found in Tables 1 2 3 4 5 .
Theme 1: Experience of Stress Responses Due to Personal and External Factors Throughout the fire and evacuation, women experienced varied stress responses during and/or following the event, including significant emotional reactions and lack of sleep. "The fire is all around us. It's like we were watching a movie or were stuck in a nightmare. It's like our whole world is falling down on us. We can feel the heat from the fire at our house and ash is falling from the sky. We're all in shock and can't move." [P165]. Several women also experienced pregnancy complications potentially related to heightened stress such as intrauterine growth restriction, C-section, and preterm births. Acts of kindness as sources of support. "The workers...were all staring at me and felt so sorry this was happening while I was in labor. They were very nice and offered us their rooms, and even offered to take our pets and look after them for the night." "Two guys asked us if we needed help with our bags. We told them we didn't have a ticket + were on standby. They immediately handed both of us their tickets, then helped us carry our bags to the plane. Whoever these two strangers are...thank you, from the bottom of my heart." Provision of needs as sources of support. "We had so many offers for places to stay, food, and even the doctor that checked the baby offered us to stay with her family!" "People were helping us in any way they could! A kind man in Spruce Grove took my mom and sister out shopping, and a sweet old lady handed us money after hearing our story. I was in awe of all the compassion." "My OBGYN finally explained to me that extreme stress can cause a baby to stop growing. There are medical terms that I researched and googled like crazy. Intrauterine growth restriction, placental insufficiency, small for gestational age." [P228]. The women also shared stressors and fears related to the event included separation from family members or significant others; fear of losing their homes; fears of losing possessions; fears of running out of gas; waiting in traffic; and well-being of their baby. These stressors and fears often highlighted the women's values, such as connection with family, safety of their children, and protection of their homes, representing uncontrollable circumstances external to the individual. "We headed down the one way on the wrong side of the road towards the camps. The highway was gridlocked. I can remember my husband saying, "One quick shift of the wind + were all wiped out." Pure terror set in as we waited in traffic for 2 hours." [P97]. Frequently, interactions between pregnancy and the environment served as an additional barrier to evacuation as women experienced difficulties including limited physical capabilities, the need for washroom facilities, or fatigue. While not atypical to pregnancy, these limitations created additional challenges for women during evacuation. Proximity to labour added additional uncertainty and stress (see Table 1 ). Writing expressed the value of relationships and togetherness with family members and spouses or partners. Whereas separation from significant others was a source of fear and stress, reunions with loved ones eased stressful emotions (see Table 2 ). Connection and support from the community was also an important facilitator of resilience during women's experiences of the fire and evacuation. Pregnant women received assistance from service providers and health care workers who examined and offered escorts to help women reach safety quickly. The women also were offered compassion and kindness from others, with one woman and her family being allowed into a camp after it was full. One new mother with a one-day old baby shared: "Two guys asked us if we needed help with our bags. We told them we didn't have a ticket + were on standby. They immediately handed both of us their tickets, then helped us carry our bags to the plane. Whoever these two strangers are...thank you, from the bottom of my heart." [P116]. Strangers made significant personal sacrifices from allowing participants to move in front of them in traffic or giving up flight tickets to ensure safety was reached. Women experienced generosity from others through donations and offers of places to "Even though this day will always be engraved in our memories, the kindness and generosity of strangers and the true efforts of our first responders will never be forgotten. There are no words to express our gratitude and love for you all." Gratitude for others. "And to my husband; thank you for being my rock, my strength, my shoulder to lean on and my person to hug. Thank you for keeping our family safe". "I will be forever grateful to my family and my husband for keeping me calm and for getting us to safety." Thankfulness for positive outcomes. "Although being loaded on a city bus 5 hours after a c-section with newborn twins wasn't ideal -I will always be grateful for the care we were given and part of me will always wonder how much worse it might have been if I had been evacuated at 38 weeks pregnant with breech twins and gone into labor." 3.2 Circumstances were framed positively. Positive feelings toward outcomes. "We lost all of our material possessions that day but in the months to follow we were blessed far beyond what we could have imagined." "We were one of the lucky ones that didn't lose our home. I thank god every day for: keeping everything we have worked so hard for and everyone safe, for not going into labor due to all the stress, and that my fiancé was on night shift that day because without him I'm not sure how our story would've turned out." Positive feelings in difficult situations. "It wasn't the best living arrangement but it could have been worse too." "Our daughter will have a crazy birth story to tell when she gets older!" Pride in community resilience. "It is amazing to see how strong our city has been since the Beast, and the compassion we've all received...It makes me so proud to say that Fort McMurray is my home." "I am proud to say that I love the community that I live in. I will always be Fort McMurray strong." stay. Women expressed feeling cared for and overwhelmed by the community support.
Theme 3: Performance of Resilience-Enhancing Activities Women described engaging in resilience-supporting behaviours. Across the narratives, they commonly wrote of practicing gratitude and framing circumstances positively. Women expressed gratitude for those who assisted them during the evacuation as well as during labour and delivery. They were thankful to the first responders who fought the fire, and to the individuals who provided support after the event. "[T]he kindness and generosity of strangers and the true efforts of our first responders will never be forgotten. There are no words to express our gratitude and love for you all." [P165]. Women expressed gratitude for significant others and extended families who kept them grounded during the evacuation and the aftermath. Furthermore, they identified positive aspects of the experience, for example, recognizing "Our daughter will have a crazy birth story to tell when she gets older!" . Finally, the pregnant women expressed pride and solidarity with their community (see Table 3 ), reflecting on their pride in how the citizens of FMWB successfully navigated difficult circumstances.
Theme 4: The Roles of Pregnancy and Motherhood in the Experiences of Loss and Resilience The women identified disruption of transitional changes into new or unique roles, occupations, and interactions with the environment related to pregnancy as a result of the wildfire. Many of the women also noted that pregnancy was a time of excitement and anticipation, indicating "I decided that morning [May 3] to start bouncing on an exercise ball cause I was anxious to meet my little man" [P67]. Others additionally noted that ordinary routines were also required, including caring for children, household tasks, and Pregnancy is an exciting time. "We were scared as first time parents, but so excited for our new adventure." Pregnancy as a time of preparation. "May 1st -I spent the day getting all the last minute details finished. I was due to have our baby girl on May 5th so I knew there wasn't much time left to get everything done. I was organizing her nursery and I was nesting so much that I spent hours scrubbing the grout on the bathroom floor. I just wanted everything perfect for when we brought our first born home from the hospital." 4.2 Women performed mothering occupations and routines. Importance of regular routines. "I was two days shy of 36 weeks pregnant while I was making dinner for my husband + 2-year-old on the day of May 3rd when people started to voluntarily evacuate from certain areas of the city." "I started with my morning routine of getting our 4-year-old ready for preschool." Grief over the loss of life. "I hear people tell their stories of loss, loss of home, loss of items. Some have property damage. As I sit and listen to all these stories, I am weeping on the inside for the loss of my baby. No amount of insurance money or Red Cross funds will fix my loss." A loss of pregnancy/labor roles with the experience of miscarriage. 4 "I asked for no pain medications for the DNC because I wanted to feel the pain of labor." "As November 21st approaches, I can't help but dwell on this day as it would have been my due date. I would trade spots in the blink of an eye to have my home burnt down if it meant I was still pregnant." 4.4 Women experienced loss of pregnancy/ mothering occupations. Concern over abilities to provide for their babies needs. "As we learned our stay in Edmonton would be longer than planned, I got so depressed and all I could do was cry. I was stressed about not having anything for the baby." "The nurse looked at us + told us our baby girl will be here today...Besides being happy, we were scared she was early. We didn't have anything for her. Not sure what our house situation was gonna be But Ready or not, here she comes!" Disruption of new occupations related to being a mother. "I never imagined I would be changing my twin sons' dirty diapers on a city bus. That is probably one of my most vivid memories of the evacuation. I remember thinking that the first day of my babies' life shouldn't be like this" 4.5 Giving birth brought fulfillment to pregnancy role. Birth as a sense of meaning contrasting uncertainty and loss. "After 9 hours of labor & 1.5 hours of pushing our little [baby] arrived @ 3:21 PM. He was so perfect. Everything was perfect." "My family finally arrived around 12:30 PM on May 4th -which was perfect timing. My beautiful son was born at 1:32 PM." 4.6 Women adapted mothering roles and occupations. Roles and occupations were fulfilled. "We drenched towels and covered our son's car seat in an attempt to repel any possible smoke that may come into the vehicle." "May 5th up for 24 + hours I needed to rest, but priority #1 car seats for the kids, new baby clothes, diapers etc. we had nothing for the baby" "I wake up our daughter and try to calmly continue our same routine because I don't want her to be scared. I'm already scared enough for the both of us...The faint smell of smoke is in the air, so I rush to turn our central air off. I didn't want our daughter to smell it." Resourcefulness in adapting to new circumstances. "We didn't have a crib or bassinet for the baby to sleep in, so we did the only thing we could think of -made a bed for her out of a dresser drawer...Its funny how you spend so much time preparing for a baby, getting everything ready, and at the end of the day all we needed was a safe place to sleep, food to eat, we had each other, and we were safe." external employment. However, these roles and occupations were disrupted by the wildfire, as women expressed feelings of loss and disappointment regarding what they had hoped to experience as a mother and for their new baby. "We decided to drive through the night South towards Saskatoon. 3 adults, 2 dogs and a 3-day-old baby all crammed into the SUV. Definitely not how I had imagined my first days postpartum." [P107]. For new mothers, the fire and evacuation acted as a barrier to performing and savouring new mothering occupations. In addition to disappointment in expectations, the wildfire and evacuation acted as a barrier to engaging in mothering roles Women valued the concept of home. "I will never be able to put into words how grateful I am for all first responders. The fire came close, but thank you for saving our home, + our memories." "At 1030 AM on May 5, our baby BOY was born!!! And we can also say we were fortunate enough to bring him home 54 days later." 5.2 Women experienced fear and loss surrounding place of home. Loss of expected experiences. "As a new mom, I didn't get to take my baby home. I didn't get to experience that first night at home with the newborn." "I was so scared, confused and emotional. Our baby was less than a day old. We should be home." Fear of losing homes. "I could not fathom the idea of leaving our home at that moment of my broken life leaving...if I left...it was as if everything that I knew to be true was now false. But we finally left our home in the early evening on May 3rd" "We then lock the door and reluctantly left -the house we'd finally finished".
Significance of homecoming. Homecoming restored the valued environment of home. "July 1st, we came back to our home in Fort McMurray with our now 2-month-old son. I remember walking inside and going straight to his room. Everything was just how I left it. I just sat there and held him and cried. This is your room. This is our home. Welcome home, again, sweet boy." Women felt strong emotions upon coming home. "As we made our journey home on June 2nd. I was flooded with raw emotions again...sadness + anger." "July 1st finally came, and it was the best day ever! I was going home to be with my husband. The drive was quiet. The drive coming into town was full of tears. I was so happy to be home, but so sad for those who lost their homes." and occupations, which exacerbated experiences of stress, lack of control, or grief as women sought to provide for their babies' needs. The disrupted environment resulted in barriers to carrying out mothering roles and occupations during the postpartum period. A small number of women noted experiencing a miscarriage during or following the events of the fire. While it is unknown whether the fire was a cause of the miscarriage, these narratives express tragic losses amidst their experiences of the wildfire related to the lost life and roles. Despite the loss and grief, many of the narratives also related stories of birth, acceptance, homecoming, and demonstrated resilience. These stories bring a sense of restoration with women regaining valued roles, occupations and environments as amidst grief and loss many of the women gave birth, contrasting the horror in the fire and evacuation. Women exhibited resilience in adapting to high-stress environments while maintaining roles and routines of motherhood. "May 5th up for 24 + hours I needed to rest, but priority #1 car seats for the kids, new baby clothes, diapers etc. we had nothing for the baby" [P252]. They emphasized the roles of provision and protection, finding creative ways to provide for their children's needs (see Table 4 ). Theme 5: The Importance of "Home" The significance of home was an important theme that demonstrated how women valued and interacted with their environment, as they experienced feelings of fear and loss during the evacuation and experienced resolution in coming home and connecting the concept of home with memories. Mothers frequently narrated the loss of being able to bring their baby home from the hospital, after preparing to do so commenting "I was so scared, confused and emotional. Our baby was less than a day old. We should be home." [P116]. Many women experienced fear that they may not see their homes again, bringing a sense of displacement. During homecoming, women experienced strong, predominantly positive emotions, sometimes mingled with fear and anger. "July 1st finally came and it was the best day ever! I was going home to be with my husband. The drive was quiet. The drive coming into town was full of tears. I was so happy to be home, but so sad for those who lost their homes." [P66]. For many of the women, coming home seemed to be indicative of circumstances settling. Birth and homecoming often provided a sense of resolution to these events, restoring anticipated roles and routines (see Table 5 ).
DISCUSSION This study aimed to explore the experiences of women who were pregnant during or shortly following the traumatic 2016 wildfire, with a specific focus on perceived stress and resilience shown through expressive writing. Exploration of the experiences of pregnant women during and following an unexpected traumatic event may provide increased knowledge of how pregnant women experience and respond to stress. Seeking to understand factors of stress and resiliency, along with the roles and occupations important in pregnancy can inform recovery interventions and support resilience-building in prenatal populations in the future.
Factors of Stress and Resilience Despite the added complication of pregnancy, participants displayed resilient mindsets and referenced emotion-focused coping through problem solving, gratitude, asking for and accepting help, and maintaining expected roles. Participants indicated they received immense support by others in the community. It was noted in the narratives that they believed this occurred due to their pregnancy. This finding warrants further exploration into the protective factors of community resilience, particularly in terms of prevention of long-term impacts resulting from PNMS. Connection with friends and family, emergency personnel, and supportive community members provided a buffer from elements of stress.
Roles and Occupations The person, occupation, and environment all impact a mother's ability to participate in roles and occupations important in motherhood, with stress and self-efficacy being critical to the transition (19) . Roles and occupations in pregnancy, birth, and motherhood were important themes that emerged in women's descriptions of the Fort McMurray wildfire, with women noting that the fire acted as a barrier to fulfilling mothering roles and occupations. During a natural disaster, elements within the person, occupation, and environment are subject to disruption and unpredictability, challenging a mother's role participation and occupational performance. Experiencing stress and trauma during pregnancy impact a mother's roles and occupations. As such, understanding the change or loss in roles and occupations are critical for supporting women following unexpected trauma during pregnancy.
Self-Selected Intervention The act of writing has been shown to help individuals process traumatic events. In this study, we examined the writings that women shared about their experiences of the evacuation from Fort McMurray and re-entry into the community. While it was impossible to connect with the women who chose to contribute to the book, it is possible to infer from their written text that they used this opportunity to share their thoughts and feelings about their experiences regarding their pregnancies and they were impacted by the wildfire. Through writing, they were also able to explore both the difficult emotions and situations caused by fire and evacuation as well as hopeful and positive outcomes, providing an outlet to process their experiences. Writing may have also provided the women with an outlet to explore important roles, environments, and occupations in pregnancy and birth and to process grief.
Recommendations Building resilience in pregnant women prior to experiencing trauma is one approach to reducing the harmful impacts of prenatal maternal stress. Prenatal education may provide an opportunity to teach expectant parents resilience strategies that can be drawn upon during a traumatic event. The FMWB wildfire created a stressful and unpredictable context in which pregnant women experienced significant stress responses. Community and familial support in transitioning roles and routines as disasters unfold is vital to ensuring pregnant women experience as minimal disruption as possible, and this support was frequently documented in the women's narratives. Coping mechanisms including planning or asking for help were discussed in several narratives, however, physiological stress management behaviors, including breathing or grounding exercises, were rarely discussed. While lack of inclusion does not confirm that these behaviors were not accessed, increased focus on stress management methods may be beneficial during prenatal education. This recommendation supports women through changes during the perinatal period in addition to unexpected stressful or traumatic events. Tragea et al. (36) found significant decreases in perceived levels of stress following implementation of a six-week stress management program for prenatal women, indicating that such strategies may act as a protective factor against harmful effects of stress during pregnancy. An increased ability to cope with stress may help in building resilience in pregnant women. Because natural disasters and other forms of traumatic stress are often unexpected, education on stress management may be beneficial as a widespread intervention for women during the prenatal period. Given the negative effects of stress on birth and childhood outcomes, it is important to support adaptive and healthy ways of responding to stress to build resilience in mothers. Education and health promotion on adaptive coping styles may be beneficial for prenatal women who may experience stress during pregnancy.
Areas for Future Research While the risks for women and children facing prenatal stress and trauma are well documented, more research is needed to identify effective interventions to support populations who have experienced an environmental disaster. Current research indicates PNMS impacts childhood development, demonstrating the need for research into proactive resilience-building is critical to assist pregnant mothers with managing stressors and offset potential risks to their infants. Additionally, as women who have experienced a traumatic event may face challenges due to effects of prenatal stress, such as difficulty in adapting to the role of motherhood, research in this area is of critical importance.
Limitations There were several limitations to this research study. The data used was not collected by the research team, leaving many aspects of data collection unknown, including when women wrote the narratives following the wildfire. The data contains a small sample size, representing a small number of evacuees. Participants were self-selected, as individuals volunteered to contribute to the writing project. Furthermore, due to the anonymity, researchers were unable to seek participant feedback after analysis was completed. As a result, the findings cannot be generalized to all women who were pregnant during the Fort McMurray Wood Buffalo wildfire.
CONCLUSION Experiencing a natural disaster significantly influences an individual's occupational participation and their ability to recover from the associated trauma. Pregnant women have unique barriers that may negatively impact them during a natural disaster or other forms of stressful event. Support to pregnant women could be offered through assistance with role transition and enabling mothering occupations and routines. Education on stress management and adaptive coping along with facilitating exploration and processing of traumatic experiences through writing interventions can also be used to build resiliency and aid in recovery from traumatic events. TABLE 1 | 1 Theme 1: Experience of stress responses due to personal and external factors. Themes Findings and supporting quotations 1.1 Stress Responses were unique to the individual, but a shared experience.
TABLE 2 | 2 Theme 2: Social support was a facilitator of resilience.Significant others as a source of support. "I closed the door and instantly started crying. I was 8 months pregnant. I had no idea what to pack so I took a step back and thought to myself...I need to call my husband so he can come home and help me." "My husband helped me through each day as I battled my emotions. He was simply amazing <3 But it's still hard." Connection with loved ones as a stress reducer. "As soon as I seen my sister we both started to cry, I was happy to be somewhere safe and with family." "[My husband] abandoned his vehicle and walked the rest of the way to us through the thick smoke. When I finally saw him walking through the smoky parking lot at the hospital, it was better than my wedding day. I could breathe again. I felt safe. With flames all around us I wasn't scared. I knew he would keep us safe." Themes Findings and supporting quotations 2.1 Support from significant others facilitated resilient attitudes. 2.2 Support from others/ community facilitated resilient outcomes.
TABLE 3 | 3 Theme 3: Performance of resilience enhancing activities.Thankfulness for hospital staff, first responders, and other service providers. "Thank you from the bottom of my heart to the emergency staff at the [hospital], especially the PICU ward, and the respiratory staff for helping our sweet boy in his fight" "The kitchen was filled with staff working overtime trying to ensure everyone got a good meal...-thank -you [company name]!" "Thank you to the emergency crews for leaving your families to save our city." "We would like to also thank all the emergency personnel from all around Canada for helping our city when we needed it most." Gratitude for kindness from strangers. "I get out of my SUV and ask the guy behind me. Please can my husband jump ahead of you, I'm worried being 3 days overdue we need to be together. He said "of course". Thank you whoever you are -the man from Tower Rd hauling your camper." Themes Findings and supporting quotations 3.1 Gratitude and thankfulness were expressed.
TABLE 4 | 4 Theme 4: The roles of pregnancy and motherhood in the experiences of loss and resilience. Themes Findings and supporting quotations 4.1 Pregnancy was a time of anticipation.
Feelings of loss regarding the expectations of new motherhood. "As a new mom, I didn't get to take my baby home. I didn't get to experience that first night at home with the newborn." "We decided to drive through the night South toward Saskatoon. 3 adults, 2 dogs and a 3-day-old baby all crammed into the SUV. Definitely not how I had imagined my first days postpartum." Feelings of grief for perceived failure in fulfilling their role as a mother. "I feel guilty. I couldn't protect my beautiful little baby girl and I failed her when she needed me most to be strong and fine and to hold it together despite everything going on around us. When people make comments about her size, although she is growing and completely healthy, but still petite for her age, I am reactive because it reminds me of my first failure to protect her and keep her safe. My first experiences as a mother are guilt and helplessness that I could not do a better job in protecting her." .3 Women experienced loss/ disappointment in relation to roles of being pregnancy/ mother.
TABLE 5 | 5 Theme 5: Place of home was important in women's experiences. Themes Findings and supporting quotations 5.1 The concept of home was valued.
Frontiers in Public Health | www.frontiersin.org
May 2022 | Volume 10 | Article 725256
|
10.3389/fsoc.2023.1063613
|
cc-by
| null | null |
openalex
|
Much of the research on platform workers has focused on individuals involved in low-skilled and highly standardized tasks. However, platform workers are not a homogeneous group. Utilizing a classification system that makes a distinction between di erent layers of platform control and grouping platforms according to how they divide decision rights between platforms and workers, we examine how and for what purposes platform workers operating in three types of control contexts have practiced and developed their digital agency for making out. The study, based on an analysis of platform webpages and semi-structured interviews of food couriers, freelancers, and interim managers, shows that workers can exercise their digital agency on all three types of platforms, but di erent platforms create di erent conditions for this depending on their special forms of control. In addition, the forms of control also a ect to what extent workers are motivated to direct their agency for making out. Instead of regarding platform work as just another layer of a periphery segment in the labor market, our analysis suggests that platforms exercising algorithmic control are new types of arenas for work, which seem to reproduce, or even amplify, the inequalities found in the o ine world of work in the digital world.. Introduction Autonomy and control have been key concepts in sociological studies analyzing the labor process. In labor process theory, a theoretical framework inspired by Braverman's (1974) seminal work, employers' need for control and workers' struggle for autonomy have been examined through Marx's well-known distinction between labor and labor power. Autonomy and control have also been important topics in mainstream sociology of work. In his classic ethnographic study, Roy (1954) depicts how manufacturing workers strive to maintain a counter-system of control for "making out" in response to managers' efforts to narrow their shopfloor autonomy. The issues of autonomy and control have played an important role in experiments inspired by sociotechnical systems theory as well. In fact, the idea of a (semi-)autonomous group has been the most common (re)design feature of sociotechnical projects promoting quality of work and industrial democracy (Guest et al., 2022) . Computerization has changed the context of sociological debate on autonomy and control in work, and the Internet has become the basis of a globally available information space, and a new sphere of social action and for interconnecting people. Much of the research has analyzed such development as a digital revolution of work, which-besides requiring new skills-enables new forms of managerial surveillance (e.g., Briken et al., 2017; Zuboff, 2019; Moore and Woodcock, 2021) . Like workers in all previous stages of industrial history, also workers in the digital environments can develop strategies of "making out" for securing higher earnings, clearing up space for autonomy in work, or fiddling the management system for their own benefit. However, workers' acts of "making do" vs. "making out, " i.e., workers' exercise of their labor power with vs. against the grain of corporate intentions, are often intertwined and difficult to disentangle, containing elements of both consent and resistance (McCabe, 2014) . Platform workers are an example of a new group of workers whose work is largely managed via smart technologies, such as algorithmic allocation of tasks, monitoring and tracking devices, and ranking and rating tools. The focus of sociological research has been on new forms of algorithmic control and their putative degrading effects on work, information asymmetries between workers and platforms, and the lack of safety nets in typical platform work without the benefits and protection provided by standard employment relationship (e.g., Bergvall-Kåreborn and Howcroft, 2014; Stewart and Stanford, 2017; Lehdonvirta, 2018) . At the same time, less attention has been paid to the new skills and coping strategies that workers can develop as their knowledge of the operation and logic of the control systems grows. Working with such systems can help workers also to cope with other kinds of digital work environments for the future. This paper combines conventional sociological research interest in autonomy and control in work with research interests deriving from educational studies on digital agency (Passey et al., 2018) and communications studies on digital inequality (Helsper, 2012; Van Deursen et al., 2017) . We explore in Finnish context the ways in which workers on three types of labor platforms seek to maintain and increase their autonomy and opportunities for making out. The paper contributes to existing research in three main ways. First, it uses "digital agency"-a concept developed in educational sciences but repurposed for our sociological inquiryas a framework for analyzing and contextualizing platform workers' coping activity. In this way, the paper aims to increase the conceptual understanding of the workers' opportunities to practice agency in platform work. Second, the paper contributes to existing research on the autonomy/control nexus in platform work by analyzing the workers' agency in a context where control is viewed as a multi-layered phenomenon. By doing so, it aims to shed light on the role of the platforms' control structures for factors enabling vs. constraining the workers' conditions for practicing agency. Finally, the paper brings similarities and differences in the opportunities to practice agency between workers operating via three types of platforms into a systematic comparison with each other, thereby illustrating the reproductive nature of the platforms for social inequalities in the labor market. Choosing three different types of platforms for the analysis enables a versatile parallel examination of the wide spectrum of platform control structures. Although the share of platform workers among all employed people in developed industrial countries is still relatively small, we consider it important to better understand the logic of labor platforms and its implications for the workers' autonomy, agency, and coping strategies. Platform companies are forerunners in algorithmic management, but it is expected that its applications will increasingly spread to more traditional forms of work, having a major impact across the economy (Fourcade and Healy, 2017; Zuboff, 2019; Gilbert and Thomas, 2021) . The COVID-19 crisis also gave an extra boost to platform companies, many of which profited from the crisis and conquered markets from more traditional players (Schaupp, 2022) . With the help of our paper, we want to draw attention to the multifaceted nature of platform work and its control structures, as well as their importance for the future quality of working life, social inequalities in the digital labor market, the needs for and possibilities of the regulation of platform work, and new potential areas for academic studies. The article is organized as follows. The next section elaborates the research questions and research tasks and the underlying theoretical discussions. This is followed by a description of the data and methods. Thereafter, the results are presented. Finally, the results, contributions, and limitations of the study are discussed.
. Theoretical foundations, research question and research tasks Scholars have developed different classifications of labor platforms as well as different images to describe and analyze platform work (Vallas and Schor, 2020) . However, much of the research on platform workers has focused on individuals involved in low-skilled tasks, such as food couriers, ride-hail drivers, or micro-taskers, as showcases of precarious "digital labor." At the same time, those doing more demanding knowledge work via online freelancing platforms have been the subject of less sociological studies (however, see Schörpf et al., 2017; Nemkova et al., 2019; Sutherland et al., 2020; Rahman, 2021; Seppänen et al., 2021; Wood and Lehdonvirta, 2022) . A recent study in Denmark found out that, of all platform workers in the country, about one third were "established workers" many of whom were able to combine platform work with high-skilled and well-paid jobs in the conventional labor market (Kristiansen et al., 2022) . Given the fact that platform work is a multifaceted phenomenon, also the autonomy/control nexus can take different forms in such work. In the following, we would like to emphasize the importance of three aspects that are particularly relevant from the perspective of our paper.
Separation of power and control First, in platform work that is performed outside standard employment relationship, the context of the autonomy/control nexus differs from that of typical work performed in such a relationship. Labor platforms' modes of control and organization differ in this respect, not only from traditional organizational hierarchies, but also from markets and (collaborative) networks (Aneesh, 2009; Kellogg et al., 2020) . Platforms delegate and distribute control among the participants-including clients and workers-but, at the same time, they centralize power (Vallas and Schor, 2020) . In other words, a discussion of autonomy and control in platform work reaches only to a limited extent the elements of power associated with such work. From the perspective of our paper, this means that by analyzing different platform control structures, we cannot draw straightforward conclusions regarding the distribution of power between the different parties on different types of platforms.
The dual nature of control The observation of control as delegated and distributed leads to our second aspect. Platform workers face dual control that comes from the direction of the platform on the one hand and the direction of the client on the other. The specific forms of control and their weighting between these two sources may greatly vary between platforms. This duality, which finds its mathematical embodiment in the platform's algorithmic management, can be a major concern for the workers' reputation and access to new tasks, depending on the specific characteristics of the platform's control system (e.g., Schörpf et al., 2017; Rahman, 2021; Wood and Lehdonvirta, 2022) . Another duality of control in platform work is its simultaneous technological and normative basis. Gandini (2019) talks about techno-normative control that platforms set upon workers. Techno-normative control is articulated in two main ways: on the one hand, client-led practices that rank and rate workers, and, on the other, digital monitoring and surveillance of work performances by the platforms. These two manifestations of control also limit spaces where workers can practice and develop their agency.
Control as layered The final aspect relevant to our paper concerns platform control as a layered process. Here, our main source of inspiration is Maffie's (2020) division, which distinguishes between three stages of control. The first stage (architecture of identity) covers how a platform creates a traceable identity for each platform user. In the second stage, the platform centralizes the key elements of an exchange, such as trust, communication, and payment. Finally, the platform can exercise market control and set the terms under which workers and clients enter in an exchange. Platform centralization builds on the architecture of identity and market control, in turn, on centralization. In practice, labor platforms differ greatly in how far they go in this process. We utilize the concept "digital agency" to map out special features that workers need for making out on platforms. Digital agency refers to the individual's ability to control and adapt to a digital world, comprising three elements (Passey et al., 2018) . Besides digital competence, digital agency requires digital confidence (in brief, utilizing one's digital competence in different digital domains in an agentic way) and digital accountability (in brief, utilizing one's digital competence responsibly and ethically). Digital agency can be considered transformative when workers, either individually or collectively, have a capacity to voluntarily form and implement intentions that go beyond and transform the accepted routines and given conditions of activity (Vänninen et al., 2015) . Transformative agency helps workers to maintain a sense of meaningfulness in their work and activities. Besides tangible actions, such as making do/out, it includes reflection, sensemaking, and planning. Platform workers' digital agency does not have to question the power exercised by platforms. However, digital agency gives workers the potential to deviate from, or act against, the grain of platform intentions that they perceive unfair and look for and learn about loopholes or vulnerabilities involved in the control system to pursue their own goals. Examples of this kind of activity can be found in several studies on platform workers (e.g., Shapiro, 2017; Galière, 2020; Kellogg et al., 2020; Bronowicka and Ivanova, 2021; Heiland and Schaupp, 2021; Seppänen et al., 2023) . We consider this activity through which workers can gain extra space and freedom in their work here as the core manifestation of workers' ability to make out. In that sense, making out can be considered not so much as resistance to the (control) system, but rather resistance within the system (e.g., Roy, 1954; Burawoy, 1979; McCabe, 2014; Perrig, 2021) . The research question is derived from the above theoretical discussions. The research question is how and for what purposes platform workers operating in three different types of control contexts have practiced and developed their digital agency for making out. We consider workers' agency as a particularly relevant research topic in the analyses of those working on all types of labor platforms for two reasons: first, like other independent workers, platform workers must compensate for their lack of organizational support with their own complementary personal arrangements, and second-what is special for platform workers also compared to other independent workers-platform workers operate in a new ecosystem that includes many uncertainties and opacities (Sutherland et al., 2020) . Moreover, we consider that the issue of digital agency is of crucial importance for platform workers, even if their actual work contains physical performances, because many key stages of the task allocation/acquisition and the actual work process are technologically mediated and algorithmically controlled (Curchod et al., 2020; Kellogg et al., 2020 ). To answer the research question, we set ourselves two research tasks. First, we examine what forms of control there are on different labor platforms and how and to what extent the platforms differ from each other. The analysis utilizes Maffie's (2020) classification. Its advantages, from the perspective of our paper, are that it is one of the few classifications that makes a distinction between different layers of platform control and explicitly groups platforms based on how they divide decisions rights between platforms and workers. Based on the classification, we distinguish six dimensions of control of which performance evaluation, centralization of workerclient communication, and centralization of payment demonstrate how platforms centralize exchange. Filtering, price setting, and matching, in turn, are analyzed as aspects of market control exercised by platforms. We assume that workers can exercise their digital agency on all types of platforms, but different platforms create different conditions for this depending on their special forms of control (Sutherland et al., 2020; Veen et al., 2020) . Our assumption is that the more work performance can be standardized, the less room it leaves for worker agency and, in this way, constrains workers' chances of making out. This assumption is also in line with the stratification hypothesis presented in communications research. The hypothesis suggests that the digital world of work reproduces existing social inequalities by replicating offline structures and because offline human capital carries over to the digital world (Van Deursen et al., 2017) . Second, we examine how workers have practiced their digital agency in different platform control contexts. Different contexts may give rise to different manifestations of digital agency depending on, among others, how fair workers consider them to be. The fairness of platform work practices is an important and rising research theme (e.g., Faullant et al., 2017; Graham and Woodcock, 2018; Fieseler et al., 2019; Pfeiffer and Kawalec, 2020; Shanahan and Smith, 2021; Seppänen et al., 2023) , but here we only touch upon it as part of answering to our research question. We assume that the less fair workers perceive platform operations, the greater motivation they have to practice and develop their digital agency for making out.
. Materials and methods We selected three types of platforms for this study on the grounds that they mediate different types of work and assuming that the nature of the mediated work is associated with the forms of platform control and through this to workers' use of their digital agency. The selection was preceded by an analysis of webpages and terms of service of 46 labor platforms operating in Finland, where they were examined whether their human resource management follows more market vs. corporation logics (Immonen, 2023) . In that study, the platforms were divided into six models based on the emergence and characteristics of those logics and the variations between them. The grouping provided us with foreknowledge of platform forms of control. The type descriptions in Subsection 4.1 were formed based on a combination of an analysis of webpages and interview data using Maffie's (2020) terminology. As the first type, we selected food-delivery platforms that convey standardized work requiring low skills, represented in our material by Alpha and Beta (pseudonyms). The second type in the study is a global marketplace Gamma (a pseudonym). The tasks mediated through Gamma can be characterized as skilled knowledge work performed by professionals. The third type is a smaller Finnish platform Delta (a pseudonym) specialized in interim management. The main sources of information in this study were semistructured interviews with platform workers and an analysis of the platform webpages. Interview participants were identified in two main ways in connection with two research projects. Freelancers working via Gamma were selected among respondents to an online survey who indicated that they were also willing to be interviewed. Food couriers and experts working via Delta were acquired in collaboration with the companies. Invitation letters prepared by the researchers were sent through the companies' information systems, and workers contacted the researchers directly using the contact information provided in the invitation. At every stage, it was underlined to the workers that the companies were not involved in the research and did not know who had signed up for the interviews. The first registrants were selected to be interviewed. The total number of interviewees was 32. The interviews were conducted at different times depending on the platform. All 10 food couriers were interviewed in 2021 and 2022 via Teams or phone. The length of the interviews varied from 50 to 90 min. Nine of the interviewees were men, and eight of them had an immigrant background. Eight of the couriers handled deliveries by car, one by bike, and one by both, depending on the situation. Half of the couriers had experience working via both platforms. At the time of the interview, three worked only for Alpha and four only for Beta. The interviewees differed greatly in status and platform activity. Five of them also had full-time or part-time employment elsewhere, three were students, and only two were full-time selfemployed. Four worked through the platform daily, four weekly such as weekends, and 2 monthly or less frequently. The Gamma material included 15 interviews. The interviews were conducted in 2018 and 2019, lasting 40-80 min. One of the interviews was conducted face-to-face and the others via Skype. Most interviewees were men (N = 11) and native Finnish speakers (N = 10). Here, also, the interviewees formed a heterogeneous group in knowledge area, status, and platform activity. The biggest occupational categories were translators (N = 4), graphic designers (N = 3), and consultants (N = 3). Seven of the professionals were full-time self-employed, whereas five were in salaried employment elsewhere, two were students, and one was unemployed. Three worked through the platform daily, five weekly, four monthly, and three less frequently. Seven people who had experience working through Delta were interviewed in 2021 and 2022 via Teams. The interviews lasted 45-90 min. All interviewed were native Finns, five of them men. All the interviewees had an academic degree and long-term experience working in various business management tasks, also as salaried employees. They all had own business name, but it was not uncommon that their recent careers included alternating periods as interim and salaried managers or other high-level specialists. At the time of the interview, two were in employment relationship to a company. Their typical assignments were long, spanning months. One of the authors conducted all interviews of persons working through Gamma and for whom Alpha was the main platform, and another author all other interviews. The interview protocol was initially modified from a study by the Institute for the Future (Anderson and Westberg, 2016) . It started with mapping of the workers' basic background information and the significance of platform work for them. Thereafter, the interview form was divided into themes, including the platform operations and workers tasks, transactions, interaction/communication, control of and support in work, problems, freedom of action, learning opportunities, fairness, and the role of society. Each main theme was further divided into subthemes and guiding questions. The questions were in each case modified to better address company-specific factors. The interviews were recorded and transcribed with the permission of the interviewees, and the transcripts were used in the coding process. The interview data analysis proceeded as follows: First, the interview data were roughly coded using the ATLAS.ti software by three of the authors into three categories of agency by naming them "expressions of tangible actions, " "reactions to problematic issues, " and "the future." The third category was inspired by Emirbayer and Mische's (1998) article on agency. In all three categories, the focus was on agency that expresses the relation between the worker and the platform. The analysis method can be called abductive (Paavola, 2021) , meaning that both empirical insights from the data and theories of agency (see above) were used in the analysis. Each interview was first coded by one of the authors and thereafter discussed together. After several rounds, one author checked all final encodings. Thereafter, citations/episodes demonstrating the three categories were recoded into "questioning, " "making do/out, " and "ideating." "Questioning" included criticism of, resistance to, reflecting on, or questioning actual problematic issues. Questioning can be considered a trigger for taking other agentic actions. Making do and making out are action-oriented types of agency, where the interviewees tell about something they do, have done, or are planning to do in the future. However, the episodes in this category differ from mere declaratory speech through the deep feelings, commitment, or desire involved that indicate how the content is relevant or important for the interviewees. We first looked at acts of making do/out as one category before making a more fine-grained distinction between them in the analysis. "Ideating" looked at the interviewees' future developmental intentions in their work contexts. It was conceptualized as suggested improvements to other people, to the platform, or to other organizations or institutions. Together, the three categories of questioning, acting (making do/out), and ideating form a model of potential steps of changing or transforming the normal course of work activities (Heikkilä and Seppänen, 2014; Vänninen et al., 2015) . However, because of the diversity of the topics involved in agentic episodes around the worker-platform relation, we do not know whether, to what extent, or how the issues of questioning turn into actions or ideating. The categorization only shows us the qualities of agency in the data. Finally, the analysis was concluded in two parts: as an overview of the three categories of digital agency and a more detailed examination of acts of making do/out in the three types. In all categories, the prefix "digital" was considered in a broad way and all agency taking place in the platform environment was included. This is justified, as algorithmic tools and systems significantly mediate and shape workers' possibilities for practicing agency on the platforms.
. Results In the following, we present our results in three subsections. We first present the division of platforms into three types based on Maffie's (2020) classification. Next, we examine what forms of worker agency were found in the interview text material. Finally, we give examples of episodes of making out in the data.
. . Autonomy and control on platform labor exchanges The results of the first research task, which focused on the forms of control on different labor platforms, are summarized in Table 1 . As was our initial assumption, the platforms clearly differ from each other in many of the dimensions of Maffie's (2020) classification, forming three distinctive types; the two food-delivery platforms operate on largely similar principles. In the following, the types are described in more detail. Type 1. The two food-delivery platforms represent the first type. Both control the number of workers registered on the platforms, but neither has explicit eligibility criteria for becoming a courier. Exchange on the platforms takes place as an interaction between clients, couriers, shops or restaurants, and the platform. Platforms' control structures also affect the autonomy of shops and restaurants in many ways, but, here, the focus is exclusively on couriers. Both platforms have performance evaluation systems for workers based on client reviews and data collection on work performances. In Alpha, both client ratings and the data collected by the platform affect the couriers' inner rankings within the platform, affecting their possibilities to get new work shifts. Couriers are ranked every 2 weeks in a way that determines the order in which they can book shifts for the forthcoming days, i.e., those ranked highest are first in line. Beta used to have a similar procedure, but it changed the system so that couriers can now sign in to work whenever they want. Common to both platforms is that it is not completely clear for workers how client reviews and the collected data are used in the platforms' algorithmic management procedures and task allocation. Beta's policy is more transparent, and it has informed couriers that their availability for performing the task and their distance from the pick-up place are the only influencing factors in task allocation. Beta also collects data on work performances but for monitoring potential neglects related to deliveries and not for ranking couriers. Communication on both platforms is centralized, leaving in normal cases only little need for interaction between clients and couriers. Clients and couriers can communicate by phone only in the final stages of the delivery when food or product is brought to clients. In other stages, communication is supposed to take place via the platforms' help desks. Both platforms use forced matching where they exclusively set the rules of work allocation and allocate tasks to potential workers by using algorithmic management. Couriers operate anonymously, and clients cannot influence who handles the order. Both platforms also exclusively determine consumer prices and the level of compensation for couriers of made deliveries. In both cases, it is up to the platforms to decide through algorithmic management how many and what kind of tasks workers receive, directly affecting workers' earnings. Both platforms use economic nudges to match supply with demand, in addition to which Alpha directly controls supply through its shift booking system. Beta pays all couriers only for made deliveries, whereas Alpha applies two types of contracts. Some couriers have old contracts that guarantee them a basic salary for made hours added with payment for made deliveries, whereas recently joined couriers are paid only based on the number of deliveries. The overwhelming platform control over matching and price setting means that both platforms do not act only as market creators, but they also have an undisputed role as market regulators (Maffie, 2020) . Type 2. The second type is represented by Gamma, a global marketplace where clients can search and hire skilled freelancers The cornerstone of Gamma's control system is its rating procedure that enables clients and workers to evaluate each other after the project ended. Clients' five-scale ratings form the basis to a worker's score, which is visible to all clients and on which her/his online reputation is based. Those with highest ratings and the thereby achieved badge showing success have an advantage on the platform when competing for new assignments. Besides the score, filtering is affected by other things, such as freelancer's activity on the platform, the number of projects completed, and possible competence certificates. Gamma offers workers different options to attach to the platform. In normal cases, Gamma limits the number of projects to which workers can apply in a month but by paying an extra fee workers can raise the number. While matching occurs quite freely without the platform intervening, communication between clients and workers is closely supervised by Gamma. Messaging between clients and workers is monitored by filtering expressions that may involve contact information. The reason for strictly controlling transactions between the parties is that payments must be managed through the platform and the service fee taken by the platform is tied to a freelancer's earnings from a client. Gamma does not control work performances as such, but in hourly-paid projects it allows clients to supervise working through an app that takes screenshots of the worker's desktop every few minutes. Such a technological monitoring is intended both to enable clients to supervise working and to certify workers' right to the agreed fee. Gamma does not directly restrict workers' autonomy by participating in job design or determining workers' price requests or levels of compensation. Negotiation on terms of the projects is left to clients and workers. Type 3. Delta represents the third type. Unlike the previous types, the platform does not control actual working in any way but has an important role in work allocation. Delta does not have formal eligibility criteria, and basically anyone can register as an expert suitable for demanding management tasks. However, before experts are allowed to present their profiles with named references and apply for projects on the platform, Delta checks the authenticity of the information. The projects are client-determined and often require high-level expertise and extensive experience from workers. Thus, workers often have great autonomy in their projects, but-unlike freelancers in Gamma-the nature of the tasks also require occasional onsite working. The platform's role is emphasized in work allocation, which distinguishes this type from the two others. Matching is neither forced nor open, but it can be characterized as single-sided. In this type of matching, all registered experts can apply for a project, but the platform screens the applicants and decides which of them will be presented to the client anonymized. In this way, the platform maintains extensive power over who will have access to a project. The purpose of anonymization is to prevent discrimination and to contribute to the fact that the selection takes place purely based on the merits of the applicants. During matching, communication between worker and client is centralized, as in the other types, meaning that negotiations between clients and workers are also managed through the platform. Terms of working and compensation are also negotiated on a tripartite basis between clients, workers, and the platform. While work performances are not controlled by the platform, payment transactions must be handled through the platform's payment system. However, clients and workers can also sign contracts in which payments take place pass the platform, but, in this case, clients must pay a special exit fee to the platform. Compared to the previous types, in type 3 the platform plays a lesser role in the actual work process. The platform's controlling role is emphasized in matching, which requires a lot of traditional manual work from the platform staff. Single-sided matching for this type of demanding expert projects still requires human judgement and cannot be left to algorithmic decision making.
. . Digital agency on three types of platforms This subsection examines expressions of workers' digital agency in the three types. Applying the categorization presented in Section 3 to the analysis of transcribed interview text data, 488 episodes in total were found, showing a clear difference in their distribution between the types (Table 2 ). Questioning was prevalent among couriers, whereas making do/out was the most common type of expression in the two other types. Type 1. In the food-delivery data, nearly half of the episodes contained questioning, most targeting the platform. The episodes dealt with multiple issues, but, here, we briefly describe only two of them. The first concerned platform practices, focusing on the ways couriers were controlled, insufficient communication between couriers and the platforms, unexpected changes in the terms of couriers' contracts, the platforms' abstinence to encourage interaction between couriers, and inflexibility in cases of problems with weather. A discrepancy between the platforms' marketed freedom and couriers' status as independent workers, and their experiences of being controlled like employees was high on the list. The second major issue concerned competition between couriers. In Alpha, couriers are ranked according to their work performance into levels that determine their chances to choose work shifts. However, many factors affecting their performance are beyond their reach. A courier may have a long relationship with the platform, but, still, only the latest 2-week period determines her/his ranking, a fact considered unfair by many. In Beta, couriers are free to work whenever they want. This caused complaints about long waiting times and wondering about Beta's continuous recruiting of new couriers. Couriers on both platforms expressed that they do not know how tasks are allocated to them. As agentic actions to questioning issues, couriers tried to find out how matching operates by analyzing their gigs, asking questions from the platforms' support centers, or discussing with their colleagues. Acts characteristic of "making do" were found in the ways workers combine courier work with their family life, studies, or hobbies, how they use platform techniques for their benefit, and their attempts to work faster. Working faster enables them to take more assignments and earn more money, and, in Alpha, every additional assignment increases their chance of attaining a higher ranking. Here, couriers act according to platform intentions, but they still experience autonomy in doing so. Acts of "making out, " in turn, are seen in couriers' tricks or shortcuts in bending the rules of the platforms or combining working with both platforms for their own benefit (see Subsection 4.3). The focus of "ideating" was on platform practices and technologies, including in some cases also considerations of benefits for platform companies and clients. The couriers wished for more information and better visibility about algorithms in rankings, payments, and task allocations. Higher payments were also wished, but many couriers regarded that their level of earnings very much depends on themselves. Restriction to the number of couriers, greater respect for them, and the possibility to have a steady salary were typical issues on couriers' wishing list to the platforms. Type 2. Questioning issues that directly affect income from the platform-client ratings, obscure ranking system, and (global) competition over assignments-was frequent among freelancers. A major source of dissatisfaction was the high service fee taken by Gamma. As a counter strategy, freelancers developed their competence levels, sought for clients also from other sources, considered leaving the Gamma platform, or collaborated with clients outside the platform. While couriers' interaction with clients is thin and shortlived, platform practices in Gamma encourage freelancers to develop long-term relations with clients in different ways. Despite the fact that freelancers saw platform services in a relatively positive light, these also raised criticism among them. It was widely believed that platform services favor clients at the expense of freelancers, and the reciprocal rating system has the potential to sanction the reputation of freelancers much more than that of clients. Such experiences of injustice gave rise to multiple forms of gaming behavior, as will be described in the next Subsection. "Ideating" in the case of freelancers largely centered on how to diminish competition from low-cost countries and with lowerskilled freelancers on assignments, such as founding new platforms operating only in Finland. Freelancers also hoped for better possibilities for networking with their online peers and more information about clients and platform practices and rules. Type 3. Experts working via Delta also questioned the imbalance between supply and demand on the platform. There is only a limited number of assignments available for hundreds of people looking for them. The platform's practice of anonymizing applicants was also questioned as well as the platform's rather undeveloped level of technology. Unlike Gamma, which is global and maintains arm's length relationships with freelancers, Delta operates locally and organizes social events where experts and clients can meet each other. Delta's encouragement of experts' social networking was applauded, but its business impact on the platform was also questioned. Building social relations with clients and occasional rotation between platform work and employment with the same clients are typical examples of "making do" by experts. The dense social networks prevailing between the platform and many of the experts and clients raise the threshold for gaming behavior among experts, radically distinguishing type 3 from the previous two. In some cases, this may even have led to mixing of roles between the parties, such that experts have acted as advertisers of platform services to potential clients. However, manifestations of making out can also be found. At least in one case an expert had negotiated directly with a client about getting a project. This is against Delta's protocol, but it was apparently possible with its tacit agreement. The experts' development ideas did not primarily relate to how the position of those working via platforms could be enhanced, but rather how platforms could improve their (customer) service. This is understandable given their experience working in management positions. Many of the ideas also focused on the gig economy more broadly and not so much Delta. . /fsoc. .
. . Episodes of making out: Examples Here, we present typical episodes of making out found in our data. The examples are under four headings with some example quotes gathered in Table 3 (see references to them in the text). As we know from previous literature (McCabe, 2014) , and as the examples below will show, drawing a line between making do and making out is not straightforward.
. . . Working on multiple platforms Many food couriers had experience working on both platforms under study. They can switch platforms by assessing which one best serves their interests at each point of time, as illustrated by quote 1 in Table 3 . As one platform offers potentially more gigs on busy times and the other predictability, the worker strategically moves between the platforms depending on demand. This strategy involves questioning each platform's downsides. The objective of this kind of making out by using two platforms is to avoid waiting times and maximize income and work autonomy. Most interviewed experts on Delta also utilized multiple channels for marketing their services and expertise, including other platforms. The motivation for such a strategy of decentralization was both the small number of projects available via the platform and questioning of the platform's assumed operational logic. The assumption was that the platform strives to allocate projects to experts evenly, putting especially those with running projects at a disadvantage when competing for new projects. Using multiple platforms in cases like this was a making-out strategy for expanding one's possibilities to find interesting assignment and smoothing out income stream (Table 3 : quote 2). As stated above, many freelancers on Gamma also sought clients from alternative sources. Unlike experts on Delta, their main motivation for doing so was to bypass the platform's high service fee.
. . . Taking on new roles Some freelancers are not registered on Gamma only as "talents" but also as clients. This is perfectly permissible, like in cases where freelancers outsource parts of their own assignments to other freelancers. However, in some cases, freelancers' underlying motives for doing this may work against corporate intentions and be seen as acts of making out by distorting competition among freelancers. By taking on the role of "artificial" client, freelancers can strive to improve their success in the competition for assignments by getting a more in-depth view of the platform's operations from the client's point of view as well as their freelancer competitors' competences and application strategies (Table 3 : quote 3).
. . . Tricks In some cases, workers use "tricks, " i.e., practices that are not in line with the interests or rules of the platform to promote their own interests. One courier used Alpha's backpack even when gigging for Beta. The courier preferred Alpha's backpack because it was more comfortable and practical, justifying his behavior with a lack of commitment to the platforms (Table 3 : quote 4). There was also another case where a courier wore "wrong" gear for the same reason. This lack of commitment, which lowers couriers' threshold for gaming, also comes out apply in the following quote: "Every time I'm in my car I have both bags." Many of the tricks are efforts to game algorithmic management and the way it affects workers' assignments and rankings. Couriers on Alpha are automatically put on forced break if they reject three invitations in a row. However, they have learned to work around this by taking a break at an appropriate time and logging in again (Table 3 : quote 5). At Gamma, the score has a significant impact on freelancers' conditions for receiving assignments from clients, but, at the same time, their opportunities to fully influence it are limited. One common way to influence one's score is to carefully select what kind of clients and projects one takes on. This is not an act of making out as such. It is possible for a freelancer to stop the project if he/she believes that he/she would receive a bad client review. This kind of strategy mainly comes into question in hourly projects, where a freelancer has received payment for the work already done, and it cannot be used without justification and too often. A freelancer can also "just in case" give good reviews of clients to increase their chances of getting new projects from them, or knowingly "order" good reviews from acquaintances (Table 3 : quote 6).
. . . Disintermediation Taking the client outside the platform can be considered the most radical form of making out that emerged in our material. Many of the platforms' controlling practices specifically aim to prevent this (see also Zhu and Iansiti, 2019) . The business model of the platforms is based both on the service fees they receive from transactions via the platforms and on the information superiority and the consequent strengthening of their market position that grows with each new transaction in relation to workers, clients, and potential competitors. Disintermediation is not permitted on Gamma and Delta. On Gamma, some freelancers still do it in all silence while they continue working via Gamma with other clients (Table 3 : quote 7). Instead, among interviewed experts on Delta, examples of such radical acts of making out did not come up. In cases of disintermediation, the clients had paid a recruitment fee to Delta in accordance with official contract protocol.
. Discussion and conclusions Here, we first reflect on our findings regarding the different types. Thereafter, we present the key theoretical and practical contributions of the paper. Finally, we highlight the limitations of our study and the related future research needs.
. . Reflection on the findings According to our empirical analysis, in type 1 workers' acts of agency are largely oriented toward questioning. In the light of the assumptions presented in Section 2, the high level of standardization of the work process combined with workers' lack of knowledge of how the algorithm allocates tasks and ranks
Example quotes
Working on multiple platforms Quote 1: "If everything is okay with [Beta], then I go for Beta, but if there is not [work], then I go for Alpha. The problem with Beta is there is no limit, everyone can start working any time. So, it happens like tomorrow if I go at 12 o'clock, the lunch time, I won't get much task, because there are many people working for Beta. But with Alpha you have to book a shift, then they already have predicted how many tasks will come this weekend" (Alpha/Beta). Quote 2: "If a project matching to your profile becomes available, you should apply if you have the time and resources. But then again, because projects appear so infrequently and there is a large number of members on the platform, it is worthless to try even if you have time, because you will not get another from there if you have one active project running. But, of course, you can try from the outside, which is why I am also in that other service" (Delta). Taking on new roles Quote 3: "Well, it [registration also as a client] definitely gives you a sense of your competitors and what's out there. Especially if you use it from both sides as a client and a freelancer. I think it's really the key, you need to know how it works from both sides. If you don't do that, you're missing out of 50 per cent on the platform and how it works. So, using it as a client helps me understand my competitors. . . [. . . ] And that's a really huge asset for anyone who is on [Gamma] , understand what goes on there on the other side" (Gamma). Tricks Quote 4: "The relation to these companies is so distant as a courier that you do it for yourself. I don't take any responsibility to promote the company because it's not that fair. If I was a paid employee and I had professional pride and security and facilities where to see colleagues, that kind of work community, so then it would be a totally different matter" (Alpha/Beta). Quote 5: "You can trick so that there comes a gig that you don't want to take, so you can ask for a break in the app, for example a 5-min break. And if it's not busy, they accept it without. . . And then that order goes away. And you are on a break. And when that gig has gone away, you stop that break immediately, and then you're working again, and there comes new gigs again" (Alpha). Quote 6: "I asked a friend to hire me on the website, so I could have a job listed on there, and he had actual work, he needed me to look at his résumé. I said, "look, I'm gonna lose 20 percent but could you please at least hire me through Gamma so I would have the job rating, " and he said "sure." We went through the process, and then afterward, he told me how it works, explained behind the scenes what he was seeing and how many people were applying for the job" (Gamma).
Disintermediation Quote 7: "But these fixed-priced projects are entirely different things. I don't often advise clients that we shouldn't take them through [Gamma] , but I do offer them a discount if we don't. Because then we will both save money on labor costs and it's often fine for them" (Gamma). them limits in many ways their opportunities for moving from mere questioning to actual making out. Perrig (2021) talks of gamification-from-above in the context of platform work, referring to algorithmic control that gives workers enough information to participate in the game (to direct their behavior) but not enough to be able to game the system. By collecting vast amounts of data from all stages of the work process, food-delivery platforms can continuously fine-tune their algorithms-making them increasingly opaque to workers-and better direct workers' actions. Through this, the platforms can also better predict the amount of labor needed in different places at any given time and learn how to better match demand and supply with the help of behavioral and economic nudges. In this way, they can also raise the threshold for potential competitors to enter the market. Couriers play a similar game of making out as manufacturing workers described by Roy (1954) and Burawoy (1979) , but with changed rules. Manufacturing workers' gaming may have been individual, but it was supported by a well-established social organization of work. We found few examples of couriers' collective agency in our interview data. This finding also applies to forms of agency that Stewart et al. (2020) call-in the absence of workplace collectivism-workspace collectivism. In this respect, our results resemble those of Veen et al. (2020) from Australia but differ from many other studies in Europe that include examples of selforganization and collective expressions of voice (e.g., Kellogg et al., 2020; Bronowicka and Ivanova, 2021; Heiland and Schaupp, 2021; Cini, 2023) . This may be because many of the couriers in our databut also in Finland in general-come from diverse immigrant backgrounds. Alpha used to have designated teams for couriers, coordinated by salaried team leaders. However, the teams were disbanded without warning and an actual stated reason, making horizontal communication between couriers more difficult. Official communication on both food-delivery platforms now takes place only between the courier and the platform. The importance of an "appropriate" level of opacity of algorithmic control is emphasized even more in work that is not as easy to standardize (Rahman, 2021) , exemplified in our analysis by type 2. Freelancers' practicing of agency in our data was manifested more often as acts of making do/out than mere questioning or ideating compared to couriers (Table 2 ) and revolving around reputational insecurities, which might overshadow their ability to access new assignments (cf. also Schörpf et al., 2017; Rahman, 2021; Wood and Lehdonvirta, 2022) . Third-party evaluations are the basis of their online reputation, embodied in their score. However, besides public client feedback, the score is also affected by their private feedback requested by the platform; a fact that freelancers or even the clients are not necessarily aware of. The obscurity of algorithmic evaluation is further increased by the fact that freelancers do not fully know what factors are causing changes in their score. Freelancer platforms also can, like food-delivery platforms, use their constantly accumulating data reserves to increase or decrease the competition between freelancers in many ways, such as by applying filters or influencing the transaction fees. Along with the level of work standardization, couriers and freelancers are distinguished by their future perspective. The job of a courier is a dead-end job in terms of learning or advancement. Couriers may socio-demographically greatly differ from each other, but as couriers, they are completely replaceable. The work of freelancers typically offers more opportunities for learning, and in addition, unlike couriers, they form a heterogeneous group also as platform workers. Some of them largely focus on their core competencies as freelancers, while others have a more entrepreneurial orientation (Nemkova et al., 2019) . Entrepreneurial orientation can be most clearly seen in our data as a conscious strategy of some freelancers to increase their reputation, acquire regular long-term customer relationships, and, with the help of these, gradually leave the platform completely. Such a way of activity, i.e., disintermediation, can be considered perhaps the most apt example of transformative digital agency (Vänninen et al., 2015) in our data. The experts operating through Delta (type 3) form a special group of platform workers-seldom, if ever, examined in studies on platform workers-which still differs from the previous ones. Delta's control structure is more transparent, and it is based on a high-trust relationship between the platform, clients, and experts. The platform has created a clearly different relationship with experts than Gamma with freelancers because the reputation of people who have held management positions is not built on the platform's client feedback, but on their entire work history from outside the platform. The experts' financial or other dependency on the platform is also less than the others' due to their typically greater wealth and larger social networks based on their previous work history. The permissive nature of the platform's control structure leaves much room for the experts' agency. However, their need to channel their agency into gaming behavior is limited by their closer, trust-based relationship with the platform. Unlike the previous two types, the platform does not appear to them so much as a faceless machine, but, rather, as an element in their overall social network.
. . Theoretical and practical contributions Our analysis shows how workers operating via three different types of platforms have practiced their digital agency. The platforms differ in their control contexts, affecting the room they leave for workers' agency and its forms of expression. It is obvious that platform workers are not a homogeneous group (Vallas and Schor, 2020) , and platform work is not just another layer of a periphery segment in the labor market (Kristiansen et al., 2022) . Platforms exercising algorithmic control are new types of arenas for work, which, in line with earlier studies on the digital divides and inequalities (Helsper, 2012; Van Deursen et al., 2017) , seem to reproduce the inequalities-for example, in work autonomy, skills development, career advancement, and labor market vulnerabilities-found in the offline world of work in the digital world. In our data, such inequalities manifest as different forms of control among the three platform archetypes, providing different opportunities for practicing agency for different groups of workers. Showing the interplay between platform forms of control, the nature of work tasks, workers' opportunities for practicing agency, and their acts of making out, based on a comparative setting, can be considered the major contribution of this paper to existing research literature. As another contribution, the study brings out how platform work not only revolutionizes ways of working, but, as a Janus-faced phenomenon, also reproduces prevailing social relations in a new context. The differences found between food couriers, freelancers, and interim managers are largely mirror images of the differences that have already existed between groups of low-skilled workers, high-skilled professionals, and managers in the offline world of work with two major exceptions. The first exception is related to the new technological affordances of algorithms. The ability of the platforms to accumulate data enables them to develop their algorithmic management in a way that increases their information superiority over all kinds of platform workers. This kind of redistribution of power, based on the platforms' growing information superiority, has potentially deteriorating effects also on the position of highskilled groups in the future digital labor market. The second exception concerns the position of groups of lowskilled workers on the platforms. The power of many blue-collar workers in the labor market has traditionally been based more on their workplace communities and trade unions than in the case of professionals or other white-collar workers. However, as already implicated, the way food-delivery work is organized does not offer the couriers the same kind of "natural" social organization and the potential for collective agency as for blue-collar workers in more traditional settings. The loss of traditional collective sources of power, without new countervailing forms of organizing, has the potential to further increase their vulnerability in the labor market in relation to other groups of platform workers. From the perspective of labor policy, both findings raise questions regarding the power advantages of platforms over workers in the labor market and the cementing, or even amplifying, effects of platform work on inequalities between different groups of workers.
. . Limitations and future research ideas Economic activities are not immune to institutional context. Our analysis focused on Finland where labor markets and industrial relations are highly regulated like in the other Nordic countries. Earlier studies have shown that, in such environments, platform companies experience pressure from social partners and legislators, having implications for their freedom of action (Jesnes, 2019; Oppegaard et al., 2020) . However, we believe that the national context has not had a major impact on the results in this case. The procedures of both food-delivery platforms seem very similar to those found in earlier studies dealing with similar work in other industrial countries. Gamma also operates around the world with uniform procedures. Delta's operations are limited to Finland. However, as stated above, it is difficult to find examples in the research literature focusing on this kind of platform work in other countries. This notwithstanding, institutional factors may in different ways limit platform companies' freedom of operation in different national contexts, an area where more research is needed. Another possible factor influencing the results could have been how the interviewees were selected. It is not possible to estimate to what extent the group of interviewees corresponds statistically to the total group of people involved in platform work in Finland, because we are currently lacking such a data basis in Finland. The group of interviewees is quite diverse in each type in socio-demographic background, labor market status, and platform activity. This suggests that the material would not be clearly biased according to any single background factor. We did not examine in more detail how financially dependent workers are on the platform. Rahman (2021) has presented that platform dependence together with evaluation setbacks is connected to how experimental vs. constrained the workers' activity on the platform are. As shown in Section 3, the dependence of the interviewed workers on the earnings via the platforms probably differs clearly from one another. This may have had an impact on how individual workers have acted on the platforms. However, the subject of the study was not the workers' different behaviors in itself, but the impact of the different platform control structures on the conditions for workers' agency. In our study, the notion of digital agency was repurposed for a sociological inquiry. The analysis focused on acts of making out, which is a concept that originates from sociological studies revolving around workers' resistance toward management or the work system (e.g., Roy, 1954; Burawoy, 1979) . However, workers' agency can emerge and evolve also from other kinds of motives. While seemingly useful in our analysis in the context of platform work, our interpretation of platform workers' digital agency may have left unnoticed other more developmental aspects of platform work that uncover potentials for workers' learning complex digital ecosystems. The purpose for which platform workers practice or can practice their digital agency-beyond making outis an important topic for future research. Finally, our study is limited in the sense that we do not know enough of the underlying intentions of platform companies, which makes it increasingly difficult to draw a line between acts of making do and making out in platform work. Our understanding is obscured particularly by two factors. First, the business principles of platform companies may differ considerably from those of more traditional companies. This is aptly brought out, for example, in the characterization of Van Doorn and Badger (2021) on how platform companies capture both monetary value associated with the services they produce and more speculative and volatile types of value based on the data they generate in connection with the provision of services. The companies' algorithms-which can be considered the most authentic reflection of these principles-are beyond our reach. The opacity of algorithms to workers or any outside observers may be intentionally built in them, as already alluded to above, but it may also be due to their machine learningbased capacity or other technological features in themselves (Kellogg et al., 2020) . Second, platform companies' intentions can also be influenced by factors other than purely businessrelated considerations, such as efforts to increase their social legitimacy or to prevent increasing regulation of their operations in advance. To increase our understanding, multidisciplinary research approaches that can better combine sociological, educational, business management, and technological (data science) expertise are welcome in future studies on platform work.
Data availability statement The datasets presented in this article are not readily available because The interview text data that form the key data source for this study is not publicly available due to the privacy agreement that signed between the interviewer and the interviewee before the interview and its recording and transcription. According to the agreement, the recordings and their transcriptions collected in the studies are treated as strictly confidential and in all stages of the studies they are only for the knowledge and use of the research teams of the studies. Requests to access the datasets should be directed to tuomo.alasoini@ttl.fi. Stewart, A., and Stanford, J. (2017) . Regulating work in the gig economy: what are the options? Econ. Labour Relat. Rev. 28, 420-437. doi: 10.1177/1035304617722461 Stewart, P., Shanahan, G., and Smith, M. (2020) . Individualism and collectivism at work in an era of deindustrialization: work narratives and food delivery couriers in the platform economy. Front. Sociol. 5, 49. doi: 10.3389/fsoc.2020.00049 Sutherland, W., Jarrahi, M. H., Dunn, M., and Nelson, S. B. (2020) . Work precarity and gig literacies in online freelancing. Work Empl. Soc. 34, 457-475. doi: 10.1177/0950017019886511 Vallas, S., and Schor, J. B. (2020) . What do platforms do? Understanding the gig economy. Ann. Rev. Sociol. 46, 273-294. doi: 10.1146/annurev-soc-121919-054857 Van Deursen, A. J. A. M., Helsper, E. J., Eynon, R., and Van Dijk, J. A. G. M. (2017) . The compoundness and sequentiality of digital inequality. Int. J. Commun. 11, 452-473. Van Doorn, N., and Badger, A. (2021) . "Dual value production as key to the gig economy puzzle, " in Platform Economy Puzzles: A Multidisciplinary Perspective on Gig Work, eds J. Meijerink, G. Jansen, and V. Daskalova (Cheltenham: Edward Elgar), 123-139. doi: 10.4337 TABLE Forms of control on three types of labor platforms. All projects are performed online. Skilled freelancers all over the world can compete for the same projects unless the client wishes to restrict this for some reason. The platform also regulates the supply by limiting the number of registered freelancers in the same field of expertise. Gamma does not control the progress of actual work performance in as straightforward way as food-delivery platforms or involve in work intermediation and negotiations between clients and freelancers. Type Type Type Performance evaluation Client ratings (Alpha and Beta) and a Client ratings and a visible Score No ranking system based on them (Alpha) based on them Centralization of Yes: only limited worker-client Yes: control of worker-client Yes: control of worker screening and worker-client communication communication messaging worker-client negotiations Centralization of payment Yes Yes Yes Filtering to clients No Yes No Price setting Platform-determined Negotiated between worker and client Negotiated between worker and client Matching Forced Open Single-sided from various fields of expertise, like design, marketing, engineering, coding, or translation.
TABLE The number of episodes of digital agency in three types of labor platforms. Type Type Type Questioning 111 (49%) 85 (42%) 14 (24%) Making do/out 69 (31%) 95 (47%) 31 (53%) Ideating 48 (19%) 22 (11%) 13 (22%) In total 228 (100%) 202 (100%) 58 (100%)
TABLE Example quotes of four types of making out.
Frontiers in Sociology frontiersin.org
|